Previous Article | Next Article 
Journal of Bacteriology, January 2001, p. 687-699, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.687-699.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Gene Cluster of Rhodothermus marinus
High-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, a
caa3-Type Oxidase Belonging to the Superfamily
of Heme-Copper Oxidases
Margarida
Santana,
Manuela M.
Pereira,
Nuno P.
Elias,
Cláudio M.
Soares, and
Miguel
Teixeira*
Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal
Received 2 June 2000/Accepted 15 October 2000
 |
ABSTRACT |
The respiratory chain of the thermohalophilic bacterium
Rhodothermus marinus contains an oxygen reductase, which
uses HiPIP (high potential iron-sulfur protein) as an electron donor.
The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I,
III, and IV (named rcoxA to rcoxD) are found in
this order and seemed to be organized in an operon of at least five
genes with a terminator structure a few nucleotides downstream of
rcoxD. Examination of the amino acid sequence of the Rcox
subunits shows that the subunits of the R. marinus enzyme
have homology to the corresponding subunits of oxidases belonging to
the superfamily of heme-copper oxidases. RcoxB has the conserved
histidines involved in binding the binuclear center and the low-spin
heme. All of the residues proposed to be involved in proton transfer
channels are conserved, with the exception of the key glutamate residue of the D-channel (E278, Paracoccus
denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine
(Y) residue and the hydroxyl group of the following serine (S) may
functionally substitute the glutamate carboxyl in proton transfer.
RcoxA has an additional sequence for heme C binding, after the
CuA domain, that is characteristic of
caa3 oxidases belonging to the superfamily.
Homology modeling of the structure of this cytochrome domain of subunit
II shows no marked electrostatic character, especially around the heme
edge region, suggesting that the interaction with a redox partner is
not of an electrostatic nature. This observation is analyzed in
relation to the electron donor for this caa3
oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which
uses an iron-sulfur protein as an electron donor, is structurally
related to the caa3 class of heme-copper cytochrome c oxidases. The data are discussed in the
framework of the evolution of oxidases within the superfamily of
heme-copper oxidases.
| |
INTRODUCTION |
In mitochondria, there is a single
terminal enzyme, cytochrome c oxidase
(aa3), in the respiratory electron transport
system that couples the one-electron oxidation of cytochrome
c to the four-electron reduction of dioxygen and to the
translocation of protons across the membrane. In prokaryotes, the
respiratory electron transport system is branched to different terminal
oxidases which differ in oxygen affinity, electron donor specificity
(e.g., quinol and cytochrome c), heme type, and metal
compositions. This diversity can be partially correlated to the
variability of growth conditions, namely, the availability of energy
source and oxygen. Despite the differences, most of these terminal
oxidases belong to the superfamily of heme-copper oxidases, which
includes the mitochondrial aa3 cytochrome
c oxidase (19, 82). However, bacterial and archaeal oxidases possess fewer protein subunits (generally three or
four) than their mitochondrial counterpart.
The presence of a subunit homologous to the largest subunit of
mammalian cytochrome c oxidase, subunit I, identifies the
members of the superfamily. Subunit I contains a binuclear center,
consisting of a heme and a copper ion (CuB) and a low-spin
heme, which transfers electrons to the binuclear center (30, 79,
89). The presence of two hemes and CuB is common to
all members of the superfamily of heme-copper oxidases (19,
82). Most of the prokaryote oxidases of the superfamily contain
homologous proteins to subunits II and III of mitochondria, in addition
to subunit I (69). As an example, the three-dimensional
structures of the mitochondrial enzyme (79, 80) and
Paracoccus denitrificans cytochrome
aa3 (30, 52) show that their
functional core structures are extremely similar with regard to
subunits I, II, and III. Subunit II is, however, more diverse than
subunit I, a fact that can be partially related to the type of electron
donor. In the Cox subfamily, subunit II of cytochrome c
oxidases contain a binuclear average-valence copper center,
CuA, the initial electron acceptor from reduced cytochrome
c (25), while quinol oxidases (82)
do not have this center. Nevertheless, it was possible to engineer a
CuA site in the subunit II of Escherichia coli
bo3 quinol oxidase (83). In some
cytochrome c oxidases, such as those from Thermus
thermophilus (40) and several Bacillus
species (62, 70), the C-terminal hydrophilic domain of
subunit II has an extension containing a heme C binding site. These
terminal enzymes constitute the class of caa3
and cao3 oxidases (19). As for
subunit III of members of the Cox subfamily, its function is still
controversial. Although the two-subunit enzyme is active in catalysis
as well as in proton translocation, it has been reported that subunit
III may be necessary for the correct assembling of the terminal oxidase
(23) or for its stabilization (9). In the
cbb3 cytochrome c oxidase subfamily, there are no mitochondrion-like subunits II and III. Instead, these
oxidases have two subunits, containing one and two C-type heme centers,
respectively (19).
Rhodothermus marinus is a strict aerobe and thermohalophile,
one classified as a new genus of the group of
Flexibacter-Bacteroides-Cytophaga (4). The
R. marinus respiratory chain contains several unique features (55-59), namely, a novel quinone-oxidizing
component: a multihemic bc complex, which is a functional
analogue of the canonical bc1 complex, and an
HiPIP (high potential iron-sulfur protein). The HiPIP is the electron
carrier between the multihemic bc complex and the
HiPIP:oxygen oxidoreductase, a terminal oxidase recently purified and
characterized (55, 58, 59). This oxidase, isolated with
four subunits with apparent molecular masses of 42, 35, 19, and 15 kDa
is the first example of a terminal oxygen reductase having as its
electron donor an iron-sulfur protein; it presents a higher turnover
with HiPIP as an electron carrier than with mitochondrial or R. marinus cytochrome c (58). The oxidase
contains hemes of the AS type, which have been reported only in T. thermophilus and in archaeal species
(39).
HiPIPs are small soluble proteins containing a single tetranuclear
cluster and are mainly found in purple photosynthetic bacteria (27, 45). It has been shown that HiPIPs can be oxidized
upon photoexcitation of the photochemical reaction center through a specific redox interaction with the tetraheme cytochrome c
(26, 29, 53). This finding indicates that HiPIPs have a
role in the bacterial photosynthetic electron transfer chain. However, HiPIPs may also have a role in the respiratory oxidative processes, as
first suggested by Pereira et al. (55) in R. marinus and by Hochkoeppler et al. (28) in the
facultative phototroph Rhodoferax fermentans. Recently, with
the purification of the caa3 oxidase and of the
bc complex from R. marinus, it was possible to
demonstrate that the HiPIP is indeed an electron carrier in the
R. marinus respiratory chain, as stated above
(58). Hence, in the last few years, evidence has been
gathered indicating that HiPIP, a long-known iron-sulfur protein,
functions as an analogue of mitochondrial cytochrome c,
i.e., as an electron donor to a terminal oxygen reductase (8, 49,
58, 71).
In order to fully characterize the unique R. marinus
HiPIP:oxygen oxidoreductase, it was essential to determine its complete primary structure. We report here the cloning and sequencing of the
gene cluster encoding this enzyme. From the analysis of the deduced
amino acid sequence of the subunits and both the previous and
additional biochemical studies, we conclude that the HiPIP:oxygen oxidoreductase is clearly identified as a member of the superfamily of
heme-copper terminal oxidases of the class caa3,
in spite of the quite distinct electron donor. Some important new
structural features, supported by modeling of the three-dimensional
structure of this enzyme, are identified and are discussed in terms of
the type of electron donor, proton transfer, and evolution of
heme-copper oxidases.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
R. marinus PRQ-62B was
the source of the chromosomal DNA used to construct a lambda library in
Lambda DASHRII replacement vector (Stratagene). E. coli strains XL1-Blue MRA (P2) and XL1-Blue MRA (Stratagene) were
the host strains for the recombinant lambda phages. The first strain
was used to amplify and to screen the genomic library. XL1-Blue MRA was
used for the preparation of lysate from pure recombinant phage,
followed by 
DNA extraction. Subcloning of lambda inserts into
p-Zero-1 (Invitrogen) was performed in E. coli XL1-Blue
(Stratagene). For further subcloning in pUC18 (see below), we used
E. coli strain JM109 (Promega).
Media.
R. marinus PRQ-62B was grown in the medium
described by Degryse et al. (15) supplemented with 0.25%
yeast extract, 0.25% tryptone and 1% NaCl. Luria-Bertani (LB)
(68) and LB low-salt (pH 7.5) (32) media were
used for standard cultures of E. coli. Antibiotics were
added at the following concentrations (in µg/ml) when necessary:
ampicillin, 100; Zeocin, 50. XL1-Blue MRA (P2) and XL1-Blue MRA
(Stratagene) strains were grown in LB medium containing 0.2% (wt/vol)
maltose and 10 mM MgSO4.
Lambda stocks in SM buffer (68) were prepared from NZY
plates (15 g of agar per liter) with NZY overlays containing 7 g of agarose per liter (68).
DNA techniques.
Genomic DNA was isolated with
cetyltrimethylammonium bromide and chloroform extraction after cell
lysis with sodium dodecyl sulfate (SDS) and proteinase K digestion
(5). DNA was isolated essentially as described in
Sambrook et al. (68), after 20% polyethylene glycol-2 M
NaCl phage precipitation from 30- to 100-ml liquid lysates and
treatment with DNase (10 µg/ml) and RNase (10 µg/ml) in order to
degrade the bacterial DNA and RNA released during lysis. Plasmid DNA
was prepared using a plasmid purification kit from Qiagen. For
miniscale extractions, plasmid DNA was prepared from 2-ml
overnight-grown cultures (7). In order to quick-check the
size of E. coli plasmids, the procedure described by Akada (1) was used.
Restriction analysis of DNA was performed using restriction enzymes and
buffers from Roche Molecular Biochemicals according
to the conditions
recommended by the
manufacturer.
To prepare insert fragments for the library, genomic DNA was partially
digested by
Sau3A1 and submitted to sucrose gradient
fractionation (
68). The appropriate fractions (containing
fragments
in the range of 9 to 23 kb) were identified by agarose gel
electrophoresis
and combined. After dialysis and ethanol precipitation,
the fractions
were used in test ligations with T4 DNA ligase (Roche
Molecular
Biochemicals) to test the integrity of the
DNA
Sau3A extremities
and in ligations to
DASH
RII-
BamHI arms (Stratagene), which were
followed by packaging and
infection of XL1-Blue MRA
(P2).
Cloning of the oxidase loci.
We used the degenerate primer
5'-TGGTKBTTYGGNCAYCCNGARGTNTAY-3' and the degenerate reverse
complementary primer 5'-NGTRWACATRTGRTGNRCCCANAC-3' to amplify genomic
DNA. These primers correspond to coding regions of the conserved
sequences, established by the alignment of subunit I sequences of a
representative group of terminal oxidases. After genomic DNA
denaturation at 94°C for 5 min, conditions for the PCR were as
follows: 94°C for 30 s, 50°C for 1 min, and 74°C for 1 min
(35 cycles), followed by 1 cycle at 94°C for 30 s, 50°C for 1 min, and 74°C for 10 min.
The 174-bp reaction product of PCR was cloned in pGEM
R-T
vector (Promega) and sequenced. The
NcoI-
PstI
fragment from this cloning
was used as a homologous probe to screen the
genomic library;
labeling of this probe was performed with
[
-
32P]dCTP using the megaprime DNA labeling system
(Amersham). Lambda
plaques were transferred to nitrocellulose filters
(
68), where
DNA was fixed by baking for 2 h at 80°C
in an oven. The filters
were then prehybridized and hybridized with the
probe, at 50°C
with 6× SSC (1× SSC is 0.15 M NaCl plus 15 mM
trisodium citrate
at pH 7.0), 5× Denhardt's reagent, 0.4% SDS
(wt/vol), 20 mM NaH
2PO
4,
and 500 µg of
sonicated salmon sperm DNA per ml. Afterward, the
filters were washed
using 2× SSC and 2× SSC-0.1% SDS at the hybridization
temperature.
After isolation and digestion of DNA from screened positive phages,
Southern blot analysis (
68) were performed, allowing
us to
select and subclone the lambda insert region containing
the gene coding
for oxidase subunit I. The probes were labeled
nonradioactively with
digoxigenin-11-labeled nucleotides (DIG-11-dUTP)
using the DIG
random-primer DNA labeling system (Roche Molecular
Biochemicals).
Hybridization with DIG-labeled probes and stringency
washes were also
performed as described by Roche Molecular
Biochemicals.
The lambda insert region of interest, included in an approximately
6.4-kb
HindIII-
EcoRI fragment, was subcloned
into p-Zero-1.
Three independent fragments of this last construct,
BamHI-
SalI,
BamHI-
HindIII, and
SalI-
EcoRI, were subcloned into pUC18, yielding
plasmids pMSAN1, pMSAN2, and pMSAN3, respectively. In the cloning
procedure, dephosphorylation of plasmid vector was performed with
Schrimp alkaline phosphatase (Amersham). The method described
by
Hanahan (
24) was used to prepare and transform competent
E. coli cells and resulted in transformation efficiencies of
10
7 CFU/µg of supercoiled plasmid
DNA.
DNA sequencing and sequence analysis.
Inserts from plasmids
pMSAN1, pMSAN2, and pMSAN3 were totally sequenced using LI-COR 4200 system (MWG-Biotech). The sequence strategy is represented in Fig.
1; the nucleotide sequence of both
strands was obtained using standard pUC forward and reverse primers and
synthetic oligonucleotide primers.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 1.
Map of R. marinus PRQ-62B genes sequenced in
this work. The gene cluster includes ORF1, as well as rcoxA,
rcoxB, rcoxC, and rcoxD, the
structural genes encoding subunits II, I, III, and IV of HiPIP
oxidoreductase, respectively. A terminator structure is found after
rcoxD. ORF2 and metF (encoding
methylenetetrahydrofolate reductase) are also represented. The
restriction sites EcoRI, SalI, BamHI,
and HindIII were used for subcloning. The
nucleotide sequences of both strands were determined in each pMSAN
insert, as represented by the arrows in the sequence strategy
scheme. The PCR products used to sequence both strands across the
SalI and BamHI junctions are also shown (see
Materials and Methods).
|
|
In order to verify that no additional small fragments were generated
when cutting the recombinant DNA with
SalI and
BamHI,
we performed PCR across the junctions comprising
these restriction
sites, as indicated in Fig.
1. We used the primer
5'-GATGCCTACGAAATCCTGGTTCA-3'
and the reverse complementary
primer 5'-AGTACTCCGTGCAGAAGACCGTG-3'
to obtain a 256-bp PCR
product across the
SalI junction. This
product was sequenced
on both strands using the same primers.
Similarly, primer
5'-AACCTCAGCCCGGAGCCGATTCG-3' and the reverse
complementary
primer 5'-GAATGCGTCCGGCCGTCACCACG-3' were chosen
in order to
get a 255-bp product across the
BamHI junction. The
PCR
templates were the genomic DNA and the p-Zero-1
insert.
Sequence data were analyzed using the Genetics Computer Group (GCG)
program package provided by the Portuguese EMBnet Node.
The search for
homologous sequences was performed at the National
Center for
Biotechnology Information in the nonredundant protein
library using the
BLAST (
3) network service. Sequence alignments
were made
with the CLUSTAL X program (
77).
Protein techniques.
The HiPIP:oxygen oxidoreductase was
purified according to the method of Pereira et al. (59).
For peptide sequencing, the enzyme subunits were separated by
SDS-polyacrylamide gel electrophoresis (PAGE) performed as described by
Pereira et al. (59) and transferred to a polyvinylidene
difluoride membrane. Each transblotted sample was submitted to
N-terminal protein sequence analysis by automated Edman degradation
(17). An Applied Biosystems model 477A protein sequencer
was used.
Ionic strength dependence studies.
Cytochrome c
oxidase activity was determined following the change in absorbance of
cytochrome c at 550 nm (
550 = 28,000 M
1 cm1) at 25°C. Horse heart cytochrome
c was prereduced by the addition of solid sodium dithionite
followed by passage over a Sephadex G-10 size exclusion column.
Different ionic strengths were obtained using 2 mM Tris-HCl (pH 8) and
different concentrations of NaCl.
Homology-based modeling.
A preliminary three-dimensional
model for subunit I of R. marinus oxidase was previously
described (59). In the present work, the composite
structure of subunit I and the truncated subunit II were modeled. The
cytochrome oxidases from P. denitrificans (52)
(PDB code 1AR1) and bovine (95) (PDB code 2OCC) were used
for the derivation of subunit I. Note that the new bovine structure was
used, since it was refined at a higher resolution. The first 20 residues and the last 41 residues of subunit I were excluded from the
model due to the lack of homology with known structures. The derivation
of subunit II is more complex, since this subunit, compared with known
cytochrome oxidases, is not as conserved as subunit I. The
transmembrane region shows a very low similarity with the cytochrome
c oxidases and part of the sequence, corresponding to a loop
in the CuA center region, is not present in the R. marinus enzyme. In addition, this oxidase has an extra
carboxy-terminal domain, which is a C-type cytochrome domain. Thus, for
the derivation of the subunit II model, different structures were used
as templates for the different domains (Fig. 2A). For the transmembrane
domain, the P. denitrificans and bovine cytochrome oxidases
were used. For the CuA domain, the T. thermophilus ba3-type cytochrome c oxidase
CuA domain (90) (PDB code 2CUA) and the
E. coli membrane-exposed domain from the quinol oxidase with
an engineered CuA center (91) (PDB code 1CYX)
were also used. Subunits I and II (without the cytochrome domain)
constitute a homology-derived model named model B.
For the derivation of a structural model of the subunit II
carboxy-terminal domain (model C), molecules from two families
(HOMSTRAD classification [
48]) of cytochromes were used:
the
cytochrome
c and
c5 families. The
first family included cytochrome
c2 from
Rhodopseudomonas viridis (
73) (PDB code
1CRY),
cytochrome
c isozyme 1 from
Saccharomyces
cerevisiae (
38) (PDB code
1YCC)
and cytochrome
c from
Thunnus alalunga (
76) (PDB
code
5CYT).
The members of the
c5 family used
were the cytochromes
c-551 from
Pseudomonas
stutzeri (
10) (PDB code
1COR) and
Pseudomonas aeruginosa (
42) (PDB code
351C). The nonstandard use
of two
families is a consequence of an overall homology with members
of
the cytochrome
c family, whereas the region containing the
heme-binding motif is highly similar to the members of the
c5 family. In the sequence alignment (Fig.
2C), a gap was inserted
in this heme region in order to exclude the
c family
information
from this motif, which is therefore generated from the
c5 family
alone. This leads to an apparent low
homology in the sequence
alignment; however, when aligned with each
family separately,
the homology is much more evident.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 2.
(A) General topology of the caa3
terminal oxidase from R. marinus models derived in this work
and the corresponding PDB structures used. (B) Alignment used to
generate R. marinus model B: the two subunits are separated
by a space in the alignment. (C) Alignment used to generate R. marinus model C.
|
|
In addition to models B and C, a model (model A) containing the full
structure of subunits I and II was also tried. Although
there is no
homology in the junction region between the Cu
A and
the
heme domains, a small connection region was used. The template
for this
connection was the engineered quinol oxidase, which presents
a longer
carboxy terminus than the other
structures.
The program Modeller version 4 (
67) was used to generate
the various models from the X-ray structures and the alignments
presented in Fig.
2. For each model type, the program generated
several
models (from 10 to 20), and the one with the lowest value
of the
objective function was
chosen.
Nucleotide sequence accession number.
The sequence has been
deposited in the EMBL data library under accession no. AJ249578.
| |
RESULTS AND DISCUSSION |
Cloning and DNA sequence analysis.
A PCR-derived probe was
used for the isolation of the oxidase genes from a genomic library
constructed in a lambda vector (see Materials and Methods). We expected
the probe, which was designed to hybridize with a sequence region of
subunit I, to serve as a total-fit probe, since the other subunit genes
could be located in the same operon, as in many other prokaryotes
(11). Four positive recombinant phages were isolated (see
Materials and Methods). Restriction DNA analysis showed that these
recombinants have overlapping restriction patterns; the restriction
site mapping of one of these recombinants is represented in Fig. 1. The
absence of rearrangements within this recombinant lambda insert was
checked by Southern blot, using R. marinus PRQ-62B
chromosomal DNA as a control and the recombinant lambda phage DNA as a
probe (results not shown). Southern blot analysis also allowed to
select and subclone the lambda insert region containing the gene coding
for oxidase subunit I; the HindIII-EcoRI
fragment drawn in Fig. 1, which hybridizes to the PCR-derived probe,
was then cloned into p-Zero-1. Further subcloning was performed, and
the nucleotide sequence of the subcloned fragments was determined (see
Fig. 1). The 6,346-bp segment of the DNA sequence extending from a
Sau3A1 site to a HindIII site (EMBL accession
no. AJ249578) is 61.1% G+C, i.e., slightly lower than the overall base
contents of R. marinus R-10 (64.4%) and R-18 (64.7%)
(2). After translation of the sequence in the six frames,
the correct reading frames for translation were confirmed by codon
usage analysis (22). Seven open reading frames (ORFs),
located on the same DNA strand and showing a uniform codon usage
and a third-position GC bias, were considered for further analysis.
Among these ORFs, the coding sequences for cytochrome oxidase subunits
I, II, and III could be easily identified since their products have
clear homologies to the equivalent subunit proteins belonging to the
superfamily of heme-copper oxidases. The relative positions of the
structural genes for cytochrome oxidase subunits are indicated in Fig.
1; rcoxB (subunit I) and rcoxC (subunit III) are
located between rcoxA (subunit II) and rcoxD
(subunit IV).
ORF1,
rcoxA,
rcoxB,
rcoxC, and
rcoxD form, most probably, an operon, since few nucleotides
separate each coding sequence.
A putative terminator sequence for the
operon is located 23 bp
downstream of the TGA stop codon of
rcoxD. A database search on
ORF1, located upstream from 5'
rcoxA, suggests homology to the
putative protein
(GI-2984387) of
Aquifex aeolicus. In this bacterium
genome,
this putative coding sequence is located between
coxC (cytochrome
c oxidase subunit III) and
coxB
(cytochrome
c oxidase
subunit II) (
14). In some
prokaryotes, additional genes required
for the biogenesis of the
oxidase, namely, genes involved in the
biosynthesis of heme A, are
found in the same cluster as the enzyme
structural genes (see reference
78). It is therefore possible
that ORF1 product is
involved in Rcox biogenesis. Two more ORFs
were identified downstream
from
rcoxD, after the putative terminator:
ORF2 and ORF3. No
significant homology to any sequence in databases
was found for ORF2.
Database searches suggest a homology of ORF3
to
methylenetetrahydrofolate reductase, a protein that in prokaryotes
catalyzes the penultimate step in the biosynthesis of methionine
and in
mammals is required for the regeneration of the methyl
group of
methionine (for a review, see reference
43).
Putative ribosomal binding sites (RBSs), complementary to the
R. marinus 16S rRNA (
4), precede the start codon of
each
ORF, except for ORF2, which could start at position 4832 or
position
4838, due to the attribution of the putative RBS for this
frame.
Inspection for codon usage of deduced amino acid sequences
reveals
a similar codon usage for ORF1, ORF2, ORF3
(
metF), and
rcox genes
(results not shown), which
constitutes an argument for considering
these ORFs to be proper genes.
However, ORF2 is a short frame,
and it is possible that the sequence
between the
rcoxD 3' end
and the ORF3 5' end is noncoding.
Also, sequences that might represent
promoter elements are found at the
end of ORF2 and in the following
region upstream of the
metF
coding sequence. Thus, the ORF2 region
could be involved in the control
of expression of
metF.
Characteristics of cytochrome oxidase subunits.
R.
marinus HiPIP:oxygen oxidoreductase was isolated as a four-subunit
complex. Tricine SDS-PAGE of the purified oxidase indicated the
presence of four polypeptides with apparent molecular masses of 42, 35, 19, and 15 kDa (59). These values are close to the ones
estimated from the deduced Rcox amino acid sequences except for
subunits I and III. As observed for other oxidases, these subunits show
anomalous migration, due to their high hydrophobicity (51,
62). Peptide amino acid sequences obtained from subunits III and
IV argue for the functional expression of the rcox cluster, since these sequences are the same as the ones deduced from
rcox DNA. Analysis of the amino acid sequence of each
subunit and analysis of the modeled subunits (see below) reveals
important structural and functional features of the R. marinus enzyme.
RcoxA.
RcoxA (316 residues, 36 kDa) has amino acid identities
of 25 and 23% with subunit II from T. thermophilus and
Bacillus subtilis caa3 oxidases, respectively
(Table 1 and Fig.
3A). The amino acid
identity with the other subunits II presented in the Fig. 3A is even
lower (e.g., 19 and 14% identities with subunit II from
Synechocystis sp. strain PCC 6803 and bovine
aa3 oxidases, respectively). However, the
hydropathy profile shows that subunit II is similar to other homologous
bacterial subunits II, with two predicted transmembrane-spanning
segments (data not shown).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Similarities between the deduced amino acid sequences of
the four rcox gene products and homologous proteins from
other microorganisms
|
|
View larger version (151K):
[in this window]
[in a new window]
|
FIG. 3.
Alignment of deduced amino acid sequences of subunits
from R. marinus Rcox oxidase with homologous subunits from
other oxidases was performed (accession nos. are in parentheses).
Rmarinus, R. marinus; Asp, Anabaena sp. strain
PCC 7120 (Z98264); Synsp, Synechocystis sp. strain PCC 6803 (P73261, P73262, and P73263); Svulcanus, S. vulcanus
(D16254, P98054, and P50677); Ecoli, E. coli (J05492);
Bsubtilis, B. subtilis (Z99111); Tthermophilus, T. thermophilus (P98005 and Q60020); Aaeolicus, A. aeolicus (067935, 067934, and 067932); Pdenitrificans,
P. denitrificans (P98002, P08306, and P06030); Btaurus,
B. taurus (bovine) (P00396, P00404, and P00415). (A) Subunit
II. The ligands for CuA are indicated by a plus sign; the
residues proposed to interact with cytochrome c (*) and
the consensus heme attachment site CXXCH (#) are also indicated. The
residues A136 to P161 referred in the text are marked with an "x"
(note that the mature P. denitrificans subunit II
starts at Q29). (B) Subunit I. The conserved histidine residues
indicated by an asterisk; the E278 residue of the D-channel
and the YS motif are also marked (#). Also marked are the residues
proposed to interact with cytochrome c ("+"; see the
text). For both subunits, identical residues are shaded.
|
|
The amino acid residues, which are the ligands for Cu
A, are
conserved in all of the cytochrome oxidases presented in Fig.
3A. In
the case of
E. coli bo3 quinol oxidase, the
Cu
A center
is absent (
12). The Cu
A
domain of
R. marinus subunit II presents
a substantial loop
deletion corresponding to residues A
136 to P
161
of subunit II from
P. denitrificans (see Fig.
2B,
3A,
and also
4). At least
one of the residues from this loop (D
159) is important for
the interaction with
P. denitrificans cytochrome
c (see below).
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 4.
Comparison between the modeled structure (model B) and
the structures of the known cytochrome c oxidases. All
structures are within the same relative orientation. The stereo
pictures were produced with GRASP (50). The
potential values in the molecular surfaces range from  10 to +10 kT/e. (A) Fold of the
R. marinus oxidase, with the hemes (in purple) and the
copper atoms (in blue). The red arrow marks the region of subunit I
where a loop deletion is observed; the green arrow marks the region of
the loop deletion in subunit II (see text). (B, C, and D). Molecular
surfaces of the oxidases colored by electrostatic potential: R. marinus (B), P. denitrificans (C), and bovine (D)
cytochrome c oxidase.
|
|
In the carboxy-terminal region of
R. marinus
HiPIP:oxygen oxidoreductase subunit II there is a cytochrome C
domain containing
a consensus heme attachment site, CXXCH. This motif
is also present
in the
B. subtilis and in
T. thermophilus caa3 oxidases (Fig.
3A), which are
described as cytochrome
c oxidases (
40,
70).
The 35-kDa subunit of the oxidase isolated from
R. marinus
contains
a heme C, as shown by heme staining upon SDS-PAGE
(
59), and
its molecular mass is in agreement with the
expected value for
the RcoxA
product.
It is proposed that subunit II is threaded through the membrane by use
of the general prokaryotic secretory pathway (sec)
(for a review, see
reference
16), in which the first hydrophobic
domain
is recognized as a signal sequence. The existence of subunit
II
N-terminal signal sequences has been already demonstrated for
several
subunits II of various oxidases, namely, those from
P. denitrificans and
B. subtilis (
6,
75).
R. marinus HiPIP:oxygen
oxidoreductase subunit II might
also have an N-terminal signal
sequence, with a cleavage site located
between positions 45 and
46 (VGA-MT). Signal sequences are
approximately 20 amino acids
in length and contain positively charged
residues in the N terminus
preceding a hydrophobic core. Some primary
sequence constraints
exist at position
3 (Ala, Gly, Ser, Val, and
Ile) and
1 (Ala,
Gly, and Ser) relative to the cleavage site, with
alanine found
especially frequently (
84). Note that the
rcoxA gene contains
at least two putative start codons:
AUG at position 951 and CUG
at position 1029. A 19-residue signal
peptide, in the range of
sizes usually found for prokaryotic signal
peptides, could be
obtained using the second codon, suggesting that
translation starts
at the CUG codon (EMBL accession no.
AJ249578).
RcoxB.
The rcoxB gene encodes a subunit of 565 residues with a molecular mass of 62 kDa which shows homology with
subunit I of cytochrome oxidases. A hydropathy plot (not shown)
suggests the presence of 12 transmembrane segments, as is the case for
the subunit I of mitochondrial (79) and P. denitrificans (30) enzymes.
Among the Rcox subunits, subunits I shows the highest degree of
identity with homologous oxidases subunits (39 and 40% amino
acid
identities with
Synechocystis sp. strain PCC 6803 and
Anabaena sp. strain PCC 7120 subunit I of cytochrome
oxidases, respectively)
(Table
1, Fig.
3B). Six totally conserved
histidine residues
(indicated by asterisks in Fig.
3B) are the ligands
of the cytochrome
a3-Cu
B center and
cytochrome
a of
RcoxB.
Proton transfer pathways in subunit I were proposed based on sequence
analysis and site-directed mutagenesis studies and were
later
corroborated by the crystal structures of the
aa3 oxidases
from
P. denitrificans and from bovine heart (
30,
79).
Pathways
D and K are named according to the conserved residues in each
channel: aspartate and lysine, respectively (
20,
31,
46).
All residues proposed to be important for proton transfer in the
channels are conserved in
R. marinus oxidase, with the
exception
of the key glutamate residue of the D-channel
(E
278,
P. denitrificans numbering). Other
oxidases in Fig.
3B lack
this glutamate residue, i.e., those from
Anabaena sp. strain PCC
7120,
Synechocystis sp.
strain PCC 6803,
Synechococcus vulcanus,
T. thermophilus (
caa3) and
A. aeolicus (COXA1).
RcoxC.
The rcoxc gene is predicted to encode a
226-amino-acid protein (26 kDa). The hydropathy profile shows that
RcoxC is a membrane protein with five predicted transmembrane segments
(not shown). N-terminal sequencing was performed on this subunit.
However, no information could be obtained on the sequence of the first residues. This fact could be due either to N-terminal blockage or
degradation of subunit III. Nevertheless, a stretch of peptide sequence
(HPPYLQH) matches the deduced amino acid sequence, confirming the
expression of this subunit. The protein presents 30% amino acid
identity with the Anabaena sp. strain PCC 7120 cytochrome oxidase subunit III and 26% amino acid identity with that of
Synechocystis sp. strain PCC 6803 (Table 1). This percentage
is lower for the caa3 oxidases from B. subtilis (19%) and T. thermophilus (21%) and even
smaller for the aa3 enzyme from P. denitrificans and COXC from A. aeolicus (17%).
However, in terms of amino acid similarity, the conservation is higher
than for subunit II; subunit III has 50 and 47% amino acid
similarities with the Anabaena sp. strain PCC 7120 and
S. vulcanus corresponding subunits, respectively, whereas subunit II has 31 and 32% similarities.
RcoxD.
The presence of a fourth subunit in bacterial
cytochrome oxidases was demonstrated for the P. denitrificans aa3 oxidase crystallized complex
(30). Also, Bacillus PS3
caa3 oxidase (74), B. subtilis aa3 quinol oxidase (37), and
E. coli bo3 quinol oxidase (33, 41,
47) are composed of four subunits. It was proposed that E. coli CyoD subunit IV is essential for CuB binding to
subunit I, being a domain-specific molecular chaperone in the oxidase complex (65, 66). R. marinus caa3
oxidase has also a fourth subunit encoded by rcoxD, a
122-residue protein (13 kDa). The N-terminal sequence of this subunit
A-H-A-T-H-H-I-I-P-R is identical to that predicted from the respective
gene, except that the initial methionine is missing. It is then
definitely demonstrated that subunits III and IV of the purified
HiPIP:oxygen oxidoreductase and RcoxC and RcoxD are the same
proteins. Subunit IV shows only 13 and 5% amino acid identities with
subunit IV from the E. coli bo3 and the
B. subtilis caa3 oxidases, respectively
(Table 1). Although these values are low, the three subunits present a
similar hydropathy profile with three hydrophobic segments (data not shown).
Analysis of the modeled structures: implications for electron and
proton transfer mechanisms.
In this work, three models of R. marinus caa3 enzyme were derived. The results for
model A (data not shown) clearly show that the available experimental
data is not sufficient to determine the complete structure, since for
each run a significantly different orientation of the cytochrome domain
in relation with the rest of the molecule was obtained. In contrast to
model A, model B is well defined (Fig. 4A). The structure is similar
overall to that of the cytochrome c oxidases, but some
important differences are found. A major difference is located in
subunit I, in the D-proton channel. As stated above, with the exception
of the E278 (P. denitrificans numbering) of
the D-channel, all of the postulated important residues of the proton
channels K and D are conserved in the R. marinus
oxidase. Mutants obtained by substitution of E278 are
unable to complete reduction of oxygen and consequently unable to
translocate protons (20, 31, 46, 64, 88). However, the
R. marinus caa3 oxidase performs the complete
reduction of molecular oxygen to water (59) and is indeed
a proton pump with the expected stoichiometry of 1 H+/e (60). The carboxyl group of
E278, which is located at the end of the channel, close to
the catalytic center, is substituted in the same spatial position by
the hydroxyl group of a tyrosine residue (Y256) in the
R. marinus enzyme (Fig. 5).
Another relevant difference in the D-channel is a glycine-to-serine
substitution in the position close to the binuclear catalytic center.
Based on theoretical calculations and Fourier transform infrared (FTIR)
spectroscopy, it has been suggested that the glutamate residue acts as
a proton shuttle, assuming two distinct conformations ("in" and
"out"), related by a 180° rotation of the carboxylate group
(61). Interestingly, in the R. marinus oxidase,
the spatial positions equivalent to the in and out conformations of the
glutamate side chain are occupied by the hydroxyl groups of the
above-mentioned tyrosine and serine residues, Y256 and
S257, respectively. Although the actual role of these
residues has still to be established, both may be active elements in
the proton pathway, either by maintaining an ordered chain of water
molecules in the cavity or by being directly involved as part of the
proton wire, where their OH groups could work in a hopping mechanism for proton transfer, assuring the proton connectivity between the end
of the D-channel and the binuclear center. Additionally, a possible
ionization of Y256 may allow a mechanism of coupling
between proton and electron capture and release.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 5.
D-channel in P. denitrificans (A) and
R. marinus (B). The heme groups from subunit I, the
CuB center, the histidine ligands, and the residues
corresponding to the D-channel in both proteins are represented.
Glu-278 in P. denitrificans and Tyr-256 and Ser-257 in
R. marinus are labeled in panels A and B, respectively.
|
|
The electrostatic characteristics of the model B of
R. marinus oxidase and the cytochrome
c oxidases are
compared in Fig.
4B, C, and D. These surfaces seem to be very similar,
but
R. marinus is closer to
P. denitrificans
than to the structure from the bovine
enzyme due to the existence of a
similar positive potential area
in the surface of the molecule, which
should be facing the cytoplasm.
If the cytochrome domain is excluded,
on the opposite side of
the molecule, a simple reasoning would imply
that the protein
should behave as a cytochrome
c oxidase,
with negatively charged
areas on the exposed copper center side,
leading to a favorable
interaction with positively charged areas, e.g.,
the heme edge
of a mitochondrion-type cytochrome
c. However,
a more careful
analysis of the residues considered to be important for
the interaction
of
P. denitrificans oxidase and
cytochrome
c (
36,
92-94) shows
that not all
are conserved in
R. marinus caa3 oxidase. In
subunit
II, specifically in the Cu
A domain, a long loop
(A
136 to P
161,
P. denitrificans
numbering) is missing (see Fig.
4A, segment
deletion, marked with a
green arrow). The absence of this loop
results in a reduced distance
between the Cu
A center and the surface
of this domain and
also in the absence of one important residue
for interaction with
cytochrome
c (D
159 in
P. denitrificans). Also in this region, D
135, which seems
to be one of the most important residues for cytochrome
c
binding (
92), is substituted by a threonine residue (see
Fig.
3A). E
126 (
P. denitrificans
numbering), D
178, and W
121, with this
last considered essential for electron transfer
(
93),
are nevertheless conserved. In subunit I,
D
156 is absent (segment deletion, marked with a red arrow
in Fig.
4A), but D
257 is present (see also Fig.
3B).
Therefore, in the absence of the
cytochrome domain, it could be
considered that part of the cytochrome
c binding site is
present in this
caa3 oxidase. However, the
cytochrome
domain present in this oxidase may change the properties for
cytochrome
c binding, in this case by reducing the
efficiency of cytochrome
c in transferring electrons to the
oxidase (see reference
58).
In order to study the cytochrome domain, model C (Fig.
6A) was created. The model is well
defined, although less accurate
in two loop structures (an upper loop
and a lower loop) surrounding
the propionate groups of the heme, since
these are the places
where the
c and
c5 families are most different (data not shown).
In the model these two loops are long, while in the
c and
c5 families
only the lower loop or the upper
loop is long, respectively. The
modeled cytochrome domain does not show
a marked electrostatic
character (see Fig.
6B and C), especially around
the heme edge
region, which is in general one of the most exposed
regions involved
in electron transfer. This is true for the cytochrome
c family,
for example, where the heme edge has been
suggested to be involved
in the electron transfer with flavodoxins
(
13,
72,
86),
cytochrome
b5
(
87), cytochrome
c peroxidase
(
54), and plastocyanin
(
81). In the actual
case of the mitochondrion-type cytochrome
c, the electron
donor to cytochrome
c oxidases, the heme edge
has long been
considered to be the entry and exit route for electrons.
The lysine
residues shown to be involved (
18,
63) in complex
formation with cytochrome
c oxidases are located around this
edge.
These positively charged residues of cytochrome
c
interact with
a number of important acidic residues located in subunits
I and
II at the surface of the oxidase, as mentioned above (
36,
92,
94). In the case of
R. marinus oxidase, if the
cytochrome domain
of the oxidase exposes the heme edge, its interaction
with a redox
partner will not be electrostatic in character, since this
region
is rather neutral (Fig.
6B and C). Therefore, molecules similar
to mitochondrial cytochrome
c would not form stable
complexes.
In fact, and in contrast to what is observed for the bovine
and
P. denitrificans aa3 oxidases,
R. marinus cytochrome
c oxidase
activity decreases with
the increase of ionic strength (Fig.
7),
i.e., it does not follow a bell-shape curve (
92). For
those
enzymes, the very low activity at low ionic strength was
attributed
to the formation of a stable electrostatic complex. A
behavior
similar to that of the
R. marinus enzyme was
observed for the
ba3 oxidase from
T. thermophilus (
21). Unlike the mitochondrial
cytochrome
c family, which presents surfaces with positive
electrostatic
potential character, HiPIPs can have very different
electrostatic
characteristics. This suggests that in HiPIPs
electron transfer
is less dependent on electrostatic interactions and
explains the
higher electron transfer rate observed with HiPIP
compared to
the one observed for cytochrome
c
(
58). A relevant example of
such behavior is the
photosynthetic reaction center of
Rubrivivax gelatinosus.
This center is able to receive electrons from both
a cytochrome
c and an HiPIP, showing a remarkable preference for
the
latter electron donor. Mutagenesis studies provide evidence
of an
electrostatic-interaction-driven binding of cytochrome
c to
the reaction center, while for HiPIP the binding is driven
by
hydrophobic interactions. Nevertheless, the binding regions
overlap in
the edge of the lower redox potential heme (
53).
Like this
photosynthetic reaction center, the HiPIP interaction
with the
cytochrome domain of the
caa3 oxidase of
R. marinus may
be mediated by hydrophobic interactions
since, whatever the proposed
orientation in the three-dimensional
structure of the cytochrome
domain, this does not show a marked
electrostatic character.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 6.
(A) Fold of the modeled structure of the cytochrome
domain from R. marinus (model C). The stereo picture was
produced with Molscript (34) and Raster 3D
(44). (B and C) Fold and molecular surfaces (same
orientation) of the cytochrome domain colored by electrostatic
potential. The stereo pictures were produced with GRASP
(50). The potential values in the molecular surface range
from 10 to +10 kT/e.
|
|
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 7.
Effect of ionic strength on the activity of the
caa3-type oxidase from R. marinus,
using horse heart cytochrome c as an electron donor, at
25°C.
|
|
Evolutionary aspects.
Subunit I from Rcox oxidoreductase
contains the conserved residues of the proposed proton channels with
the exception of the key glutamate (E278, P. denitrificans numbering) of the D-channel; homology modeling analysis suggests that tyrosine 256 and serine 257 could be the glutamate functional substitutes. Inspection of the performed sequence
alignments (see Fig. 3B) reveals that these residues are also present
in other oxidases. The correlation of these data with the 16S rRNA
bacterial phylogenetic tree shows that enzymes containing the YS motif
are present in organisms belonging to the deepest branches; on the
other hand, the glutamate-containing oxidases are only present in
purple bacteria, gram-positive bacteria, and mitochondria. These
observations suggest that the R. marinus caa3
oxidase proton pathway, presumably involving the tyrosine and serine
residues, is ancestral to the one involving the glutamate in the
gram-positive and purple bacteria and reinforces the suggestion put
forward by Castresana et al. (11) for lateral transfer of oxidase genes from gram-positive bacteria to purple bacteria, the older
proton pathway being maintained in the early divergent branches.
R. marinus HiPIP:oxygen oxidoreductase is a true
terminal oxidase (
59,
60), being the first example of a
member of the
heme-copper superfamily using a HiPIP as an electron
donor. The
electron donor specificity of heme-copper oxidases is
related
to subunit II's structure. During evolution, mutations in the
C-terminal domain would generate differences in the electron donor
substrate specificity, this evolutionary change being parallel
with the
evolution of the type of electron donor. The first electron
donor may
have been an iron-sulfur protein, since these proteins
are rather
primordial molecules (
85). If this hypothesis is
correct,
we will find
caa3 oxidases to be ambivalent with
respect
to the electron donor (in parallel with what happens with the
tetraheme cytochrome subunit bound to the photosynthetic reaction
center of
R. gelatinosus [
53]), while others
use only one type
of electron donor, i.e., an iron-sulfur protein or a
cytochrome
c. A posterior step in the evolution of the
C-terminal domain
of
caa3 oxidases would have
been its own deletion, leading to
the appearance of the
aa3 cytochrome
c oxidases.
We therefore propose that
R. marinus oxidase arose from a
common ancestor existing before the branching of the
Bacteroides,
Thermus, and
Deinococcus
genera, retaining characteristics of
this primitive bacterial
cytochrome
c oxidase. Further mutagenesis
studies on the
C-terminal domain of subunit II will be important
in order to evaluate
the binding properties and to infer the proposed
hypothesis for the
subunit II evolution of cytochrome
oxidases.
In conclusion, the study of this
R. marinus oxidase expands
the diversity of the members of the heme-copper superfamily, both
in
terms of the type of electron donor and in terms of the pathways
for
proton uptake and/or translocation. These characteristics
bring new
insight to the theory of terminal-oxidase
evolution.
| |
ACKNOWLEDGMENTS |
M. Santana and M. Pereira are recipients of grants from Praxis
XXI program (BPD/3382/96 and BPD/22054/99). This work was supported by
Project BIO/37/96 to MT.
We are grateful to P. Fernandes and I. Marques, from the
Bioinformatics Unit of the Instituto Gulbenkian de Ciência, for providing access and support in the use of GCG software for sequence analysis (Wisconsin Package). We thank Manuela Regalla for N-terminal sequencing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Tecnologia Química e Biológica, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal. Phone: 351-214469844. Fax: 351-214428766. E-mail: miguel{at}itqb.unl.pt.
| |
REFERENCES |
| 1.
|
Akada, R.
1994.
Quick-check method to test the size of Escherichia coli plasmids.
BioTechniques
17:58[Medline].
|
| 2.
|
Alfredsson, G. A.,
J. K. Kristjansson,
S. Hjorleifslottir, and K. O. Stetter.
1988.
Rhodothermus marinus, gen. nov., sp. nov., a thermophilic halophilic bacterium from submarine hot springs in Iceland.
J. Gen. Microbiol.
134:299-306.
|
| 3.
|
Altschul, S. F.,
T. L. Madden,
A. A. Schäffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipmun.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402[Abstract/Free Full Text].
|
| 4.
|
Andrésson, O. S., and O. H. Fridjónsson.
1994.
The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species.
J. Bacteriol.
176:6165-6169[Abstract/Free Full Text].
|
| 5.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl.
1994.
Current protocols in molecular biology
John Wiley & Sons, New York, N.Y.
|
| 6.
|
Bengtsson, J.,
H. Tjalsma,
C. Rivolta, and L. Hederstedt.
1999.
Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein.
J. Bacteriol.
181:685-688[Abstract/Free Full Text].
|
| 7.
|
Birnboim, H. C., and J. Doly.
1979.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Res.
7:1513-1522[Abstract/Free Full Text].
|
| 8.
|
Bonora, P.,
I. Principi,
B. Monti,
S. Ciurli,
D. Zannoni, and A. Hochkoeppler.
1999.
On the role of high-potential iron-sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs.
Biochim. Biophys. Acta
1410:51-60[Medline].
|
| 9.
|
Bratton, M. R.,
M. A. Pressler, and J. P. Hosler.
1999.
Suicide inactivation of cytochrome c oxidase: catalytic turnover in the absence of subunit III alters the active site.
Biochemistry
38:16236-16245[CrossRef][Medline].
|
| 10.
|
Cai, M.,
E. Bradford, and R. Timkovich.
1992.
Investigation of the solution conformation of cytochrome c-551 from Pseudomonas stutzeri.
Biochemistry
31:8603-8612[CrossRef][Medline].
|
| 11.
|
Castresana, J.,
M. Lübben,
M. Saraste, and D. G. Higgins.
1994.
Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.
EMBO J.
13:2516-2525[Medline].
|
| 12.
|
Chepuri, V.,
L. Lemieux,
D. C.-T. Au, and R. B. Gennis.
1990.
The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases.
J. Biol. Chem.
265:11185-11192[Abstract/Free Full Text].
|
| 13.
|
Cunha, C. A.,
M. J. Romão,
S. J. Sadeghi,
F. Valetti,
G. Gilardi, and C. M. Soares.
1999.
Modelling of electron transfer complexes between flavodoxin and c-type cytochromes.
J. Biol. Inorg. Chem.
4:360-374[CrossRef][Medline].
|
| 14.
|
Deckert, G.,
P. V. Warren,
T. Gaasterland,
W. G. Young,
A. L. Lenox,
D. E. Graham,
R. Overbeek,
M. A. Snead,
M. Keller,
M. Aujay,
R. Huber,
R. A. Feldman,
J. M. Short,
G. J. Olsen, and R. V. Swansson.
1998.
The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.
Nature
392:353-358[CrossRef][Medline].
|
| 15.
|
Degryse, E.,
N. Glansdorff, and A. Piérard.
1978.
A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus.
Arch. Microbiol.
117:189-196[CrossRef][Medline].
|
| 16.
|
Economou, A.
1999.
Following the leader: bacterial protein export through the Sec pathway.
Trends Microbiol.
7:315-320[CrossRef][Medline].
|
| 17.
|
Edman, P., and G. Begg.
1967.
A protein sequenator.
Eur. J. Biochem.
1:80-91[Medline].
|
| 18.
|
Ferguson-Miller, S.,
D. L. Brautigan, and E. Margoliash.
1978.
Definition of cytochrome c binding domains by chemical modification. III Kinetics of reaction of carboxydinitrophenyl cytochromes c with cytochrome c oxidase.
J. Biol. Chem.
253:149-159[Free Full Text].
|
| 19.
|
García-Horsman, J. A.,
B. Barquera,
J. Rumbley,
J. Ma, and R. B. Gennis.
1994.
The superfamily of heme-copper respiratory oxidases.
J. Bacteriol.
176:5587-5600[Free Full Text].
|
| 20.
|
Gennis, R. B.
1998.
Cytochrome c oxidase: one enzyme, two mechanisms?
Science
280:1712-1713[Free Full Text].
|
| 21.
|
Giuffrè, A.,
E. Forte,
G. Antonini,
E. D'Itri,
M. Brunori,
T. Soulimane, and G. Buse.
1999.
Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature.
Biochemistry
38:1057-1065[CrossRef][Medline].
|
| 22.
|
Gribskov, M.,
J. Devereux, and R. R. Burgess.
1984.
The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression.
Nucleic Acids Res.
12:539-549.
|
| 23.
|
Haltia, T.,
M. Finel,
N. Harms,
T. Nakari,
M. Raitio,
M. Wikström, and M. Saraste.
1989.
Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.
EMBO J.
8:3571-3579[Medline].
|
| 24.
|
Hanahan, D.
1983.
Studies on transformation of Escherichia coli with plasmids.
J. Mol. Biol.
166:557-580[Medline].
|
| 25.
|
Hill, B. C.
1993.
The sequence of electron carriers in the reaction of cytochrome c oxidase with oxygen.
J. Bioenerg. Biomembr.
25:115-120[CrossRef][Medline].
|
| 26.
|
Hochkoeppler, A.,
S. Ciurli,
G. Venturoli, and D. Zannoni.
1995.
The high potential iron-sulfur protein (HiPIP) from Rhodoferax fermentans is competent in photosynthetic electron transfer.
FEBS Lett.
357:70-74[CrossRef][Medline].
|
| 27.
|
Hochkoeppler, A.,
P. Kofod,
G. Ferro, and S. Ciurli.
1995.
Isolation, characterization, and functional role of the high-potential iron-sulfur protein (HiPIP) from Rhodoferax fermentans.
Arch. Biochem. Biophys.
322:313-318[CrossRef][Medline].
|
| 28.
|
Hochkoeppler, A.,
P. Kofod, and D. Zannoni.
1995.
HiPIP oxido-reductase activity in membranes from aerobically grown cells of the facultative phototroph Rhodoferax fermentans.
FEBS Lett.
375:197-200[CrossRef][Medline].
|
| 29.
|
Hochkoeppler, A.,
D. Zannoni,
S. Ciurli,
T. E. Meyer,
M. A. Cusanovich, and G. Tollin.
1996.
Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.
Proc. Natl. Acad. Sci. USA
93:6998-7002[Abstract/Free Full Text].
|
| 30.
|
Iwata, S.,
C. Ostermeier,
B. Ludwig, and H. Michel.
1995.
Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans.
Nature
376:660-669[CrossRef][Medline].
|
| 31.
|
Karpefors, M.,
P. Ädelroth,
A. Aagaard,
H. Sigurdon,
M. S. Ek, and P. Brzezinsky.
1998.
Electron-proton interactions in terminal oxidases.
Biochim. Biophys. Acta
1365:159-169[Medline].
|
| 32.
|
Kennedy, C. K.
1971.
Induction of colicin production by high temperature or inhibition of protein synthesis.
J. Bacteriol.
108:10-19[Abstract/Free Full Text].
|
| 33.
|
Kranz, R. G., and R. B. Gennis.
1983.
Immunological characterization of the cytochrome o terminal oxidase from Escherichia coli.
J. Biol. Chem.
258:10614-10621[Abstract/Free Full Text].
|
| 34.
|
Kraulis, P. J.
1991.
MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures.
J. Appl. Cryst.
24:946-950.
|
| 35.
|
Kyte, J., and R. F. Doolittle.
1982.
A simple method for displaying the hydropathic character of a protein.
J. Mol. Biol.
157:105-132[CrossRef][Medline].
|
| 36.
|
Lappalainen, P.,
N. J. Watmough,
C. Greewood, and M. Saraste.
1995.
Electron transfer between cytochrome c and the isolate CuA domain: identification of substrate-binding residues in cytochrome c oxidase.
Biochemistry
34:5824-5830[CrossRef][Medline].
|
| 37.
|
Lemma, E.,
J. Simon,
H. Schägger, and A. Kröger.
1995.
Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis.
Arch. Microbiol.
163:432-438[Medline].
|
| 38.
|
Louie, G. V., and G. D. Brayer.
1990.
High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c.
J. Mol. Biol.
214:527-555[CrossRef][Medline].
|
| 39.
|
Lübben, M., and K. Morand.
1994.
Novel prenylated hemes as cofactors of cytochrome oxidases: archaea have modified hemes A and O.
J. Biol. Chem.
269:21473-21479[Abstract/Free Full Text].
|
| 40.
|
Mather, M. W.,
P. Springer, and J. A. Fee.
1991.
Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.
J. Biol. Chem.
266:5025-5035[Abstract/Free Full Text].
|
| 41.
|
Matsushita, K.,
L. Patel, and H. R. Kaback.
1984.
Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation.
Biochemistry
23:4703-4714[CrossRef][Medline].
|
| 42.
|
Matsuura, Y.,
T. Takano, and R. E. Dickerson.
1982.
Structure of cytochrome c-551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms.
J. Mol. Biol.
156:389-409[CrossRef][Medline].
|
| 43.
|
Matthews, R. G.,
C. Sheppard, and C. Goulding.
1998.
Methylenetetrahydrofolate reductase and methionine synthase biochemistry and molecular biology.
Eur. J. Pediatr.
157:554-559.
|
| 44.
|
Merritt, E. A., and D. J. Bacon.
1997.
Raster3D photorealistic molecular graphics.
Methods Enzymol.
277:505-524[Medline].
|
| 45.
|
Meyer, T. E.,
V. Cannac,
J. Fitsch,
R. G. Bartsch,
G. Tollin, and M. A. Cusanovitch.
1990.
Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina.
Biochim. Biophys. Acta
1017:125-138[Medline].
|
| 46.
|
Mills, D. A., and S. Ferguson-Miller.
1998.
Proton uptake and release in cytochrome c oxidase: separate pathways in time and space?
Biochim. Biophys. Acta
1365:46-52[Medline].
|
| 47.
|
Minghetti, K. C.,
V. C. Goswitz,
N. E. Gabriel,
J. J. Hill,
C. Barassi,
C. D. Georgiou,
S. I. Chan, and R. B. Gennis.
1992.
Modified, large-scale purification of the cytochrome o complex of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.
Biochemistry
31:6917-6924[CrossRef][Medline].
|
| 48.
|
Mizuguchi, K.,
C. M. Deane,
T. L. Blundell, and J. P. Overington.
1998.
HOMSTRAD: a database of protein structure alignments for homologous families.
Prot. Sci.
7:2469-2471[Medline].
|
| 49.
|
Moschettini, G.,
A. Hochkoeppler,
B. Monti,
B. Benelli, and D. Zannoni.
1997.
The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells.
Arch. Microbiol.
168:302-309[CrossRef][Medline].
|
| 50.
|
Nicholls, A.
1992.
GRASP: graphical representation and analysis of surface properties.
Columbia University, New York, N.Y.
|
| 51.
|
Nicoletti, F.,
H. Witt,
B. Ludwig,
M. Brunori, and F. Malatesta.
1998.
Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes.
Biochim. Biophys. Acta
1365:393-403[Medline].
|
| 52.
|
Ostermeier, C.,
A. Harrenga,
U. Ermler, and H. Michel.
1997.
Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody Fv fragment.
Proc. Natl. Acad. Sci. USA
94:10547-10553[Abstract/Free Full Text].
|
| 53.
|
Osyczka, A.,
K. V. Nagashima,
S. Sogabe,
K. Miki,
K. Shimada, and K. Matsuura.
1999.
Comparison of the binding sites for high-potential iron-sulfur protein and cytochrome c on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center.
Biochemistry
38:15779-15790[CrossRef][Medline].
|
| 54.
|
Pelletier, H., and J. Kraut.
1992.
Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c.
Science
258:1748-1755[Abstract/Free Full Text].
|
| 55.
|
Pereira, M. M.,
A. M. Antunes,
O. C. Nunes,
M. S. Costa, and M. Teixeira.
1994.
A membrane-bound HiPIP type center in the thermohalophile Rhodothermus marinus.
FEBS Lett.
352:337-330.
|
| 56.
|
Pereira, M. M.,
J. N. Carita,
R. Anglin,
M. Saraste, and M. Teixeira.
2000.
Heme centers of Rhodothermus marinus respiratory chain. Characterization of its cbb3 oxidase.
J. Bioenerg. Biomembr.
32:143-152[CrossRef][Medline].
|
| 57.
|
Pereira, M. M.,
J. N. Carita, and M. Teixeira.
1999.
Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: a novel multihemic cytochrome bc, a new complex III.
Biochemistry
38:1268-1275[CrossRef][Medline].
|
| 58.
|
Pereira, M. M.,
J. N. Carita, and M. Teixeira.
1999.
Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: characterization of the iron-sulfur centers from the dehydrogenases and investigation of the high-potential iron-sulfur protein function by in vitro reconstitution of the respiratory chain.
Biochemistry
38:1276-1283[CrossRef][Medline].
|
| 59.
|
Pereira, M. M.,
M. Santana,
C. M. Soares,
J. Mendes,
J. N. Carita,
M. A. Carrondo,
M. Saraste, and M. Teixeira.
1999.
The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel.
Biochim. Biophys. Acta
1413:1-13[Medline].
|
| 60.
|
Pereira, M. M.,
M. L. Verkhovskaya,
M. Teixeira, and M. I. Verkhovsky.
2000.
The caa3 terminal oxidase of Rhodothermus marinus lacking the key glutamate of the D-channel is a proton pump.
Biochemistry
39:6336-6340[CrossRef][Medline].
|
| 61.
|
Pomès, R.,
G. Hummer, and M. Wikström.
1998.
Structure and dynamics of a proton shuttle in cytochrome c oxidase.
Biochim. Biophys. Acta
1365:255-260[CrossRef].
|
| 62.
|
Quirk, P. G.,
D. B. Hicks, and T. A. Krulwich.
1993.
Cloning of the cta operon from alkaliphilic Bacillus firmus OF4 and characterization of the pH-regulated cytochrome caa3 oxidase it encodes.
J. Biol. Chem.
268:678-685[Abstract/Free Full Text].
|
| 63.
|
Rieder, R., and H. R. Bosshard.
1980.
Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1 and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c.
J. Biol. Chem.
255:4732-4739[Abstract/Free Full Text].
|
| 64.
|
Riistama, S.,
G. Hummer,
A. Puustinen,
R. B. Dyer,
W. H. Woodruff, and M. Wikström.
1997.
Bound water in the proton translocation mechanism of the haem-copper oxidases.
FEBS Lett.
414:275-280[CrossRef][Medline].
|
| 65.
|
Saiki, K.,
T. Mogi,
M. Tsubaki,
H. Hori, and Y. Anraku.
1997.
Exploring subunit-subunit interactions in the Escherichia coli bo-type ubiquinol oxidase by extragenic suppressor mutation analysis.
J. Biol. Chem.
272:14721-14726[Abstract/Free Full Text].
|
| 66.
|
Saiki, K.,
H. Nakamura,
T. Mogi, and Y. Anraku.
1996.
Probing a role of subunit IV of the Escherichia coli bo-type ubiquinol oxidase by deletion and cross-linking analyses.
J. Biol. Chem.
271:15336-15340[Abstract/Free Full Text].
|
| 67.
|
Sali, A., and T. L. Blundell.
1993.
Comparative protein modelling by satisfaction of spatial restraints.
J. Mol. Biol.
234:779-815[CrossRef][Medline].
|
| 68.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 69.
|
Saraste, M.
1990.
Structural features of cytochrome oxidase.
Q. Rev. Biophys.
23:331-366[Medline].
|
| 70.
|
Saraste, M.,
T. Metso,
T. Nakari,
T. Jalli,
M. Laureus, and J. van der Oost.
1991.
The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme.
Eur. J. Biochem.
195:517-525[Medline].
|
| 71.
|
Schoepp, B.,
P. Parot,
L. Menin,
J. Gaillard,
P. Richaud, and A. Vermeglio.
1995.
In vivo participation of a high potential iron-sulfur protein as electron donor to the photochemical reaction center of Rubrivivax gelatinosus.
Biochemistry
34:11736-11742[CrossRef][Medline].
|
| 72.
|
Simondsen, R. P.,
P. C. Weber,
F. R. Salemme, and G. Tollin.
1982.
Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes.
Biochemistry
21:6366-6375[CrossRef][Medline].
|
| 73.
|
Sogabe, S., and K. Miki.
1995.
Refined crystal structure of ferrocytochrome c2 from Rhodopseudomonas viridis at 1.6 Å resolution.
J. Mol. Biol.
252:235-247[CrossRef][Medline].
|
| 74.
|
Sone, N.,
S. Shimada,
T. Ohmori,
Y. Souma,
M. Gonda, and M. Ishizuka.
1990.
A fourth subunit is present in cytochrome c oxidase from the thermophilic bacterium PS3.
FEBS Lett.
262:249-252[CrossRef][Medline].
|
| 75.
|
Steinrücke, P.,
G. C. Steffens,
G. Panskus,
G. Buse, and B. Ludwig.
1987.
Subunit II of cytochrome c oxidase from Paracoccus denitrificans. DNA sequence, gene expression and the protein.
Eur. J. Biochem.
167:431-439[Medline].
|
| 76.
|
Takano, T., and R. Dickerson.
1980.
Redox conformation changes in refined tuna cytochrome c.
Proc. Natl. Acad. Sci. USA
77:6371-6375[Abstract/Free Full Text].
|
| 77.
|
Thompson, J. D.,
T. J. Gibson,
F. Plewniak,
F. Jeanmougin, and D. G. Higgins.
1997.
The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res.
25:4876-4882[Abstract/Free Full Text].
|
| 78.
|
Thony-Meyer, L.
1997.
Biogenesis of respiratory cytochromes in bacteria.
Microb. Mol. Biol. Rev.
61:337-376[Abstract].
|
| 79.
|
Tsukihara, T.,
H. Aoyama,
E. Yamashita,
T. Tomizahi,
H. Yamaguchi,
K. Shinzawa-Itoh,
R. Nakashima,
R. Yaono, and S. Yoshikawa.
1995.
Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 Å.
Science
269:1069-1074[Abstract/Free Full Text].
|
| 80.
|
Tsukihara, T.,
H. Aoyama,
E. Yamashita,
T. Tomizaki,
H. Yamaguchi,
K. Shinzawa-Itoh,
R. Nakashima,
R. Yaono, and S. Yoshikawa.
1996.
The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å.
Science
272:1136-1144[Abstract].
|
| 81.
|
Ubbink, M., and D. S. Bendall.
1997.
Complex of plastocyanin and cytochrome c characterized by NMR chemical shift analysis.
Biochemistry
36:6326-6335[CrossRef][Medline].
|
| 82.
|
van der Oost, J.,
A. P. N. de Boer,
J.-W. L. de Gier,
W. G. Zumft,
A. H. Stouthamer, and R. J. M. van Spanning.
1994.
The haem-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.
FEMS Microbiol. Lett.
121:1-10[Medline].
|
| 83.
|
van der Oost, J.,
P. Lappalainen,
A. Musacchio,
A. Warne,
L. Lemieux,
J. Rumbley,
R. B. Gennis,
R. Aasa,
T. Pascher,
B. G. Malmstrom, et al.
1992.
Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.
EMBO J.
11:3209-3217[Medline].
|
| 84.
|
von Heijne, G.
1986.
A new method for predicting signal sequence cleavage sites.
Nucleic Acids Res.
14:4683-4690[Abstract/Free Full Text].
|
| 85.
|
Wächtershäuser, G.
1988.
Before enzymes and templates: theory of surface metabolism.
Microbiol. Rev.
52:452-484[Free Full Text].
|
| 86.
|
Weber, P. C., and G. Tollin.
1985.
Electrostatic interactions during electron transfer reactions between c-type cytochromes and flavodoxin.
J. Biol. Chem.
260:5568-5573[Abstract/Free Full Text].
|
| 87.
|
Wendoloski, J. J.,
J. B. Matthew,
P. C. Weber, and F. R. Salemme.
1987.
Molecular dynamics of a cytochrome c-cytochrome b5 electron transfer complex.
Science
238:794-797[Abstract/Free Full Text].
|
| 88.
|
Wikström, M.
1998.
Proton translocation by the respiratory haem-copper oxidases.
Biochim. Biophys. Acta
1365:185-192[CrossRef].
|
| 89.
|
Wikström, M.,
K. Krab, and M. Saraste.
1981.
Cytochrome oxidase: a synthesis
Academic Press, London, England.
|
| 90.
|
Williams, P. A.,
N. J. Blackburn,
D. Sanders,
H. Bellamy,
E. A. Stura,
J. A. Fee, and D. E. McRee.
1999.
The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 Å resolution.
Nat. Struct. Biol.
6:509-516[CrossRef][Medline].
|
| 91.
|
Wilmanns, M.,
P. Lappalainen,
M. Kelly,
E. Sauer-Eriksson, and M. Saraste.
1995.
Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.
Proc. Natl. Acad. Sci. USA
92:11955-11959[Abstract/Free Full Text].
|
| 92.
|
Witt, H.,
F. Malatesta,
F. Nicoletti,
M. Brunori, and B. Ludwig.
1998.
Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans.
Eur. J. Biochem.
251:367-373[Medline].
|
| 93.
|
Witt, H.,
F. Malatesta,
F. Nicoletti,
M. Brunori, and B. Ludwig.
1998.
Tryptophan 121 of subunit II is the electron entry site to cytochrome-c oxidase in Paracoccus denitrificans.
J. Biol. Chem.
273:5132-5136[Abstract/Free Full Text].
|
| 94.
|
Witt, H.,
V. Zickermann, and B. Ludwig.
1995.
Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c.
Biochim. Biophys. Acta
1230:74-76[Medline].
|
| 95.
|
Yoshikawa, S.,
K. Shinzawa-Itoh,
R. Nakashima,
R. Yaono,
E. Yamashita,
N. Inoue,
M. Yao,
M. J. Fei,
C. P. Libeu,
T. Mizushima,
H. Yamaguchi,
T. Tomizaki, and T. Tsukihara.
1998.
Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase.
Science
280:1723-1729[Abstract/Free Full Text].
|
Journal of Bacteriology, January 2001, p. 687-699, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.687-699.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Haveman, S. A., Holmes, D. E., Ding, Y.-H. R., Ward, J. E., DiDonato, R. J. Jr., Lovley, D. R.
(2006). c-Type Cytochromes in Pelobacter carbinolicus. Appl. Environ. Microbiol.
72: 6980-6985
[Abstract]
[Full Text]
-
Nunoura, T., Sako, Y., Wakagi, T., Uchida, A.
(2003). Regulation of the aerobic respiratory chain in the facultatively aerobic and hyperthermophilic archaeon Pyrobaculum oguniense. Microbiology
149: 673-688
[Abstract]
[Full Text]
-
Louro, R. O., Bento, I., Matias, P. M., Catarino, T., Baptista, A. M., Soares, C. M., Carrondo, M. A., Turner, D. L., Xavier, A. V.
(2001). Conformational Component in the Coupled Transfer of Multiple Electrons and Protons in a Monomeric Tetraheme Cytochrome. J. Biol. Chem.
276: 44044-44051
[Abstract]
[Full Text]
-
Osyczka, A., Nagashima, K. V. P., Sogabe, S., Miki, K., Shimada, K., Matsuura, K.
(2001). Different Mechanisms of the Binding of Soluble Electron Donors to the Photosynthetic Reaction Center of Rubrivivax gelatinosus and Blastochloris viridis. J. Biol. Chem.
276: 24108-24112
[Abstract]
[Full Text]
Top
Abstract
Introduction
