Department of Biology, Plant Science
Institute, University of Pennsylvania, Philadelphia, Pennsylvania
191041; Marmara Research Center,
Research Institute for Genetic Engineering and Biotechnology, Gebze,
Kocaeli 41470, Turkey2; and
Department of Biology, University of Bologna, Bologna 40126, Italy3
 |
INTRODUCTION |
The purple nonsulfur facultative
phototrophic bacteria of Rhodobacter species,
Rhodobacter capsulatus and its close relative Rhodobacter sphaeroides, are excellent model organisms
for studying cellular energy transduction (46). They have
versatile growth modes and are capable of both anoxygenic phototrophic
(Ps) and vigorous respiratory (Res) growth in the presence of oxygen.
They contain a photochemical reaction center (RC) (43,
44), a ubihydroquinone:cytochrome c (cyt
c) oxidoreductase (cyt bc1 complex)
(6, 45), and a c-type cytochrome as a mobile
electron carrier (7, 8), but their Ps and Res electron
transport chains are different (46, 47).
During the Ps growth of R. capsulatus, electrons are
conveyed from the cyt bc1 complex to the RC
either by the well-studied mobile electron carrier cyt
c2 (7) or the more recently
discovered membrane-anchored electron carrier cyt
cy (17), encoded by cycA and cycY, respectively. On the other hand, in R. sphaeroides, the Ps electron transport pathway can be mediated
only by a mobile electron carrier like cyt c2
(8) or its functional analogues such as isocyt
c2 (33) (Fig.
1). Although Ps-grown cells of R. sphaeroides also harbor a membrane-anchored cyt
cy, this electron carrier is not able to support
Ps growth (27). Yet the Ps growth inability of R. sphaeroides mutants lacking cyt c2 can
be restored readily by genetic introduction of R. capsulatus
cyt cy (17, 18). These findings
indicate that R. sphaeroides cyt
cy is the culprit for the Ps growth inability of
this species in the absence of cyt c2
(27), but the molecular basis of this observation remains
unknown.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 1.
Photosynthetic and respiratory electron transport
pathways of R. sphaeroides. DH, Qpool,
bc1, RC, Qox, cyt
c2, cyt cy,
aa3-Cox, and
cbb3-Cox correspond to the
respiratory NADH and succinate dehydrogenases, membrane quinone pool,
cyt bc1 complex, photochemical reaction center,
hydroquinone oxidase, soluble electron carrier cyt
c2, membrane-attached electron carrier cyt
cy, aa3-type cyt
c oxidase, and cbb3-type cyt
c oxidase, respectively. Dotted arrows highlight the
electron transfer pathways catalyzed by cyt cy.
Antimycin A (Ant A) and myxothiazol (Myx) are inhibitors of the cyt
bc1 complex.
|
|
Respiratory electron transport pathways of R. capsulatus and
R. sphaeroides are also similar but not identical
(46) (Fig. 1). Both species contain a respiratory electron
transport pathway that is branched after the quinone pool. One of these
branches contains hydroquinone oxidases (Qox) commonly
found in microbes, while the other one harbors a mitochondrial-like cyt
c oxidase(s) (Cox) converting oxygen to water
(13). While R. capsulatus contains only one
cbb3-type Cox
(cbb3-Cox) (14, 19),
both an aa3-type Cox
(aa3-Cox) (16, 36), and
a cbb3-Cox (12, 42) are
present in R. sphaeroides. In the latter species, the
cbb3-Cox is predominant in
microaerobic or Ps-grown cells, while the
aa3-Cox becomes a major component
under high O2 tension (37). In both species, the nature of the Qox is not well known. Recent works
including genome sequences (4, 11) suggest the presence of
one Qox in R. capsulatus (21, 48;
K. Zhang and F. Daldal, unpublished data) and possibly two
Qox in R. sphaeroides.
Recently, we have established that R. sphaeroides cyt
cy can function as an electron carrier between
the cyt bc1 complex and the
cbb3-Cox in R. capsulatus
(27). This finding raised the issue of whether it can also
act as an electron carrier during the Res growth of R. sphaeroides and, if so, whether it can donate electrons
to both the aa3-Cox and the
cbb3-Cox. Obviously, the presence of two c-type electron carriers and two cyt
c oxidases complicates this analysis. To facilitate
it, appropriate mutations inactivating either cyt
c2 or cyt cy were
introduced into R. sphaeroides strains lacking
either cbb3-Cox or
aa3-Cox activity. The double mutants
thus obtained converted the crisscrossed respiratory electron transport
pathways into simpler linear pathways (Fig. 1) and allowed estimation
of their efficiencies. The data demonstrated for the first time that in
R. sphaeroides either cyt c2
or cyt cy can act as an electron carrier between
the cyt bc1 complex and either the
cbb3-Cox or the
aa3-Cox. Furthermore, they indicated
that electron flow via the cyt c2 
cbb3-Cox and cyt
cy cbb3-Cox subbranches appears to be
greater under semiaerobic growth conditions. During these studies,
it was noted that in the absence of the latter respiratory pathway,
light-harvesting protein complexes, which are usually absent in the
presence of oxygen, were also produced.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Escherichia
coli strains were grown on Luria broth (LB medium) supplemented
with kanamycin, spectinomycin, or tetracycline at a final concentration
of 50, 50, or 12.5 µg per ml, respectively. R. sphaeroides strains were grown in Sistrom's minimal medium (Med A) (40) supplemented as needed with 10, 10, or 2.5 µg of spectinomycin, kanamycin, or tetracycline, respectively, per
ml. For semiaerobic growth, 2-liter flasks were filled with 1 liter of
growth medium and shaken at 150 rpm with a rotary shaker (New Brunswick
Scientific Co. Inc., Rutherford, N.J.). Photo- or chemoheterotrophic growth phenotypes and ability to catalyze the Nadi reaction
(N',N'-dimethyl-p-phenylenediamine + 
-naphthol
indophenol blue + H2O) (24) were
determined on solid media using cells grown in anaerobic jars
containing H2- and CO2-generating gas packs
(BBL) or an aerobic dark incubator at 35°C in the presence of
atmospheric oxygen. Growth rates of various strains were determined
using liquid cultures inoculated with freshly grown cells at
appropriate dilutions. As needed, cultures were first incubated
overnight at ambient temperature without shaking and then transferred
to a rotary shaker at 35°C, and the turbidity was monitored using
side-arm flasks and a Klett-Summerson colorimeter. All strains and
plasmids used are listed in Table 1.
The parental R. sphaeroides strain Ga (40)
is referred to as wild type with respect to its cytochrome profile and
its Ps and Res growth properties.
Molecular genetic techniques and construction of various R. sphaeroides mutants.
Two different insertion-deletion
alleles of cycA and cycY, carrying either
spectinomycin resistance (Sper) or kanamycin resistance
(Kanr)-conferring gene cartridges, were necessary for
constructing various double mutants lacking cyt
c2 or cyt cy and
cbb3-Cox or aa3-Cox. Thus, a
cycY::kan allele was constructed similarly to cycY::spe described previously
(27), except that a Kanr-conferring cartridge
instead of a Sper-conferring cartridge from pHP45
-K
(9) was used. The desired allele was transferred into the
suicide plasmid pSup202, conferring tetracycline resistance
(Tetr), and via the E. coli donor strain S17.1
(39) conjugated into the appropriate R. sphaeroides strains, selecting for Sper or
Kanr as required. Among the transconjugants, those that
were Tets, and hence carrying a chromosomal allele
replacement via a double-crossover event, were retained for further
analyses (3). Introduction of appropriate cycA
and cycY alleles into the R. sphaeroides
(ccoNO::kan) (MT001) and
(ctaD::spe) (JS100) mutants yielded the
(cycA::spe) (ccoNO::kan) (KD02) and
(cycY::spe)
(ccoNO::kan) (KD04) and the
(cycY::kan)
(ctaD::spe) (KD03) and
(cycA::kan)
(ctaD::spe) (KD05) double mutants,
respectively (Table 1).
Biochemical and spectroscopic techniques.
R.
sphaeroides chromatophore membranes from washed cells grown
semiaerobically in Med A were prepared using a French pressure cell at
18,000 lb/in2 and ultracentrifugation either in 50 mM
3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0)
containing 1 mM KCl, EDTA, and phenylmethylsulfonyl fluoride for
polyacrylamide gel electrophoresis (PAGE) analyses (18,
20) or in 50 mM MOPS buffer (pH 7.2) containing 5 mM MgCl2 for spectroscopic studies (50). Membrane
fragments were washed twice, resuspended at a known protein
concentration in the same buffer, and stored frozen at 
80°C until
further use. Protein content of the samples was determined by the
method of Bradford (2) or Lowry et al. (22),
using bovine serum albumin as a standard. Bacteriochlorophyll
concentration was measured by the optical absorption of
acetone-methanol (7:2, vol/vol) extracts, using an extinction
coefficient 
775 of 75 mM
1
cm1 (5). Sodium dodecyl sulfate (SDS)-PAGE
was performed using 16.5% acrylamide Tris-Tricine gels as described by
Schägger and von Jagow (35). Samples were denatured
for 5 min at 37°C in SDS loading buffer prior to electrophoresis, and
gels were stained with Coomassie brilliant blue to visualize the
polypeptides. The c-type cytochromes were revealed via the
intrinsic peroxidase activity of their heme group, using
3,3',5,5'-tetramethylbenzidine (TMBZ) and H2O2
(41). The amounts of cytochromes in chromatophore membranes were estimated by recording at room temperature reduced (with
ascorbate or dithionite)-minus-oxidized (with ferricyanide) optical
difference spectra. Either a Hitachi 3400 or Jasco 7800 spectrophotometer and the extinction coefficients
604-630 of 23 mM1 cm1,
561-575 of 22 mM1 cm1, and
551-540 of 29 mM1 cm1 were
used for a-, b-, and c-type
cytochromes, respectively.
Cyt c oxidase activities in membrane fragments were
determined using either reduced horse heart cyt c as a
substrate or by monitoring O2 consumption. In the first
case, immediately prior to the assay, horse heart cyt c was
reduced by addition of sodium dithionite, excess of which was
eliminated via chromatography through a small column containing
Sephadex G-10 as a matrix (14, 20). Oxidation of cyt
c was monitored at 550 nm in a stirred cuvette, thermostated
at 25°C, containing 50 mM Tris (pH 8.0) buffer supplemented with 0.1 mg of dodecyl maltoside per ml, 5 µM myxothiazol (Myx), and 40 µM
reduced cyt c. As needed, 2 mM potassium cyanide (KCN) was
used as an inhibitor (34). In the second case, respiratory
rates were measured by polarography at 28°C with a Clark-type oxygen
electrode (Yellow Springs Instruments Inc., Yellow Springs, Ohio).
These assays used 0.25 to 0.30 mg of membrane fragments per ml
resuspended in 50 mM MOPS buffer (pH 7.2) containing 5 mM
MgCl2, 2 mM sodium ascorbate, and 200 µM
N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)
or 50 µM horse heart cyt c (15, 48). As
needed, 100 µM KCN was used to determine the amount of
Cox-independent O2 consumption during the
assay, and enzymatic activities were expressed in moles of substrate consumed per minute per milligram of protein. TMPD was also substituted with 2,3,5,6-tetramethyl-1,4-phenylenediamine (DAD) or
3,6-dichlorophenol indophenol (DCIP) as an electron donor and KCN with
NaN3 as an inhibitor (34). Kinetic changes in
the absorption of c-type cytochromes were monitored at 551 minus 540 nm at 28°C, using a Jasco V-550 dual-wavelength
spectrophotometer equipped with a Jasco ETC-505T rapid-mixing and
temperature control apparatus (15).
To estimate amounts of the light-harvesting complexes in membrane
fragments of various R. sphaeroides mutants, 50-ml
cultures were grown aerobically with vigorous shaking in 250-ml flasks to an optical density at 630 nm of less than 0.4. Cells were collected by centrifugation and resuspended in 50 mM potassium phosphate buffer
(pH 7.0) containing 10 mM EDTA and leupeptin, pepstatin A, and Pefabloc
SC (Roche Inc., Indianapolis, Ind.) as protease inhibitors at
concentrations suggested by the supplier. After addition of 1/50 volume
of lysozyme (10 mg/ml) and 1 h of incubation on ice, cells were
frozen at 80°C for 10 min, thawed, and sonicated on ice for 5 min
at a 40% relative output in a sonicator (model 150; Dynatech
Laboratories Inc., Farmingdale, N.Y.). Cell debris was removed by
centrifugation at 13,000 rpm for 30 min at 4°C, and the supernatants
were centrifuged at 150,000 × g for 1 h. Membrane
fragments thus obtained were washed with 50 mM potassium phosphate (pH
7.0) buffer containing 1 mM EDTA, resuspended in the same buffer, and
stored at 80°C until further use. Optical absorption spectra were
recorded between the wavelengths of 380 and 1,000 nm with a Beckman
DU640 spectrophotometer, using an amount of R. sphaeroides membranes corresponding to a total protein content
of 0.2 mg per ml in 10 mM potassium phosphate (pH 7.2) buffer
containing 1 mM EDTA.
Chemicals.
All chemicals, including the redox mediators,
were of analytical grade and obtained from commercial sources as
described previously (15). n-Dodecyl
-D-maltoside was from Anatrace; horse heart cyt
c (type VI) and TMPD were from Sigma-Aldrich Chemical Co. (St. Louis, Mo.).
| |
RESULTS |
R. sphaeroides strain Ga cells grown under
semiaerobic conditions contain both
cbb3-Cox and
aa3-Cox activities.
Both
cbb3-Cox and
aa3-Cox activities in the wild-type
R. sphaeroides strain Ga can be fully inhibited by
cyanide (CN) and azide (N3)
anions. However, while CN affects both Cox
activities similarly, N3 inhibits them
differently (34). Thus, to assess whether cells grown
under semiaerobic conditions contained both of these enzymes, ascorbate/cyt c-dependent Cox activity was
measured in the presence of increasing concentration of these
inhibitors, using membranes of wild-type strain Ga (Fig.
2). The
cbb3-Cox activity was totally inhibited by 100 µM N3, whereas
the aa3-Cox activity required 2 to 3 mM N3 for full inhibition. Moreover, the
inhibition by CN was monophasic (90% inhibition at 20 µM KCN), while that by N3 was biphasic,
with a decrease in slope around 100 µM NaN3,
corresponding to about 60% inhibition of total activity. Thus, the
R. sphaeroides cells used in this study contained both
of the Cox activities simultaneously.
View larger version (10K):
[in this window]
[in a new window]
|
FIG. 2.
Inhibition of the cyt c oxidase activity
found in R. sphaeroides membranes. Inhibitory effects of
CN (circles) and N3 (triangles)
on ascorbate and cyt c oxidase activities found in membrane
fragments of R. sphaeroides wild-type strain Ga were
determined as described in Materials and Methods.
aa3 and cbb3 correspond
to the aa3-Cox and
cbb3-Cox, respectively. See the text
for details.
|
|
R. sphaeroides mutants with linear,
c-type cytochrome-dependent respiratory electron transport
pathways.
In R. sphaeroides, c-type cytochromes
containing branches of the respiratory electron transport pathways are
complicated due to the presence of the cyt c2,
cyt cy,
cbb3-Cox, and
aa3-Cox (Fig. 1). To simplify these
crisscrossed pathways, four double mutants containing only one electron
carrier (cyt c2 or cyt
cy) and one Cox
(cbb3-Cox or
aa3-Cox) were obtained (Table 1).
Growth phenotypes of these mutants confirmed that among the electron
transport components mutated, only cyt c2 was
required for Ps growth (8) (Table 2). Their doubling times under Res growth
conditions (about 100 to 120 min on Med A at 35°C) were similar to
those of their parents, with the exception of the
cbb3-Cox
aa3-Cox double mutant
(ME127), which grew slightly slower (doubling time of approximately 150 min). In particular, Res growth of the cyt bc1 (BC17), cyt
c2 cyt
cy (Gadc2cy), and
cbb3-Cox
aa3-Cox (ME127)
mutants were consistent with the presence of an alternate respiratory
pathway, involving a Qox (13). It was also
noted that mutants lacking the
cbb3-Cox (MT001, KD02, and
especially KD04) formed highly pigmented colonies under respiratory
growth conditions.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Electron transport chain components of various R. sphaeroides mutants, and their cytochrome contents in
membranesa
|
|
Next, the ability of all strains to perform the Nadi reaction
(24), described in Materials and Methods, was tested as
an indication of their Cox activities. Among them,
only the cbb3-Cox
aa3-Cox mutant (ME127)
exhibited a complete Nadi phenotype (i.e., no blue color
formation after more than 15 min of staining) (Table 2). The
cbb3-Cox mutant
(MT001) and its cyt
c2
cbb3-Cox
(KD02) and cyt cy
cbb3-Cox (KD04)
derivatives showed various degrees of Nadislow phenotypes
(i.e., formation of a bluish color within a few minutes of staining),
with KD04 being the most defective in this respect. The remaining
mutants were Nadi+ (i.e., formed a blue color in less than
1 min of staining), like the wild-type R. sphaeroides
strain Ga. These observations indicated that absence of either
the aa3-Cox (JS100), cyt
c2 (Gadc2), cyt cy
(Gadcy), or their combination (Gadc2cy, KD03, and KD05) did not affect
the Nadi phenotype. Thus, in R. sphaeroides, the
Nadi+ phenotype correlated better with the presence of an
active cbb3-Cox rather than an
aa3-Cox. It was also noted that the
Nadi phenotype of various mutants was affected by the presence or
absence of the electron carriers cyt c2 and cyt
cy and also by the growth conditions used. For
example, the cyt cy
cbb3-Cox mutant KD04
exhibited a stronger Nadi+ phenotype if younger colonies
were stained, but conversely, the cyt
c2
cbb3-Cox mutant KD02
became as Nadi as the
cbb3-Cox
aa3-Cox mutant ME127
when grown in enriched YCC medium (30).
Cytochrome contents of various R. sphaeroides
mutants.
The contents of the a-, b-, and
c-type cytochromes in chromatophore membranes, and in
membrane supernatants, of various mutants grown by respiration in Med A
were estimated using reduced-minus-oxidized optical absorption
difference spectra (Fig. 3 and Table 2).
Comparison of the reduced-minus-oxidized spectra of the
cbb3-Cox (MT001) and
cbb3-Cox
aa3-Cox (ME127)
mutants with other strains revealed significant decreases in the
amounts of the c-, b-, and a-type cytochromes
(peaks around 550, 560, and 605 nm, respectively) (Fig. 3A). In
addition, contribution of the b- and c-type cyt
subunits of the cyt bc1 complex to total cytochrome contents of membranes was readily visible in the 550- to
560-nm region when the cyt bc1
mutant BC17 was compared to other mutants. Importantly, comparison of
the cyt c2
cbb3-Cox (KD02), cyt
cy
aa3-Cox (KD03), cyt
cy
cbb3-Cox (KD04), and
cyt c2
aa3-Cox (KD05) double
mutants with their respective parents
(cbb3-Cox [MT001]
and aa3-Cox [JS100])
and with the wild-type strain Ga revealed that the 605-nm broad peak
was absent in mutants carrying ctaD::spe and
that the 550-nm peak was decreased significantly in those containing
ccoNO::kan, as expected based on their
genotypes (Fig. 3B).
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 3.
Reduced-minus-oxidized optical absorption difference
spectra of various R. sphaeroides mutants. The spectra
were recorded between 510 and 630 nm at 25°C, using membrane
fragments as described in Materials and Methods. (A) Spectra obtained
with the cyt c2 (Gadc2), cyt
cy (Gadcy), cyt
c2 cyt
cy (Gadc2cy), cyt
bc1 (BC17),
cbb3-Cox (MT001), and
cbb3-Cox
aa3-Cox (ME127)
mutants; (B) those obtained with the wild-type strain Ga and the
aa3-Cox (JS100), cyt
cy,
aa3-Cox (KD03), cyt
c2
aa3-Cox (KD05), cyt
c2
cbb3-Cox (KD02), and
cyt cy
cbb3-Cox (KD04)
mutants. Absorption peaks corresponding to the a-,
b-, and c-type cytochromes are indicated by
arrows. Note that the absorbance scale used in panel A is twice as
large as that in panel B.
|
|
In all mutants, the amount of a-type cytochromes was lower
(e.g., as low as 0.24 ± 0.01 nmol/mg of protein in the cyt
c2 cyt
cy mutant Gadc2cy) than in the
wild-type strain Ga (0.8 ± 0.04 nmol/mg of protein) (Table 2).
The b-type heme content was also low in almost all mutants
(around 1.2 ± 0.3 nmol/mg of protein); an exception was the
amount in the cyt cy
aa3-Cox (KD03) mutant,
which was similar to that in Ga (2.2 ± 0.1 nmol/mg of
protein). Further, the amounts of c-type cytochromes
dropped from 3.8 ± 0.2 nmol/mg of protein in Ga to 0.7 ± 0.02 nmol/mg of protein in cyt c2
cbb3-Cox (KD02) and
cyt cy
cbb3-Cox (KD04)
mutants as a result of mutations eliminating various c-type cytochromes. Elimination of the
cbb3-Cox decreased the total amount of the b-type cytochromes by about one-half and that of the
c-type cytochromes by more than two-thirds. Consequently,
the cyt b-to-cyt c ratios increased from 0.5 ± 0.04 in membranes of the cyt
bc1 mutant (BC17) to 1.3 ± 0.1 in those of the cyt c2
cbb3-Cox mutant
(KD02). Thus, clearly the contents of the a-, b-,
and c-type cytochromes in membrane fragments from various
strains varied as a function of the missing components.
The c-type cytochrome contents of chromatophore membrane
supernatants of the mutants were also determined using
ascorbate-reduced-minus-ferricyanide-oxidized optical difference
spectra (not shown). The data indicated that R. sphaeroides mutants carrying a cycA knockout allele
had about 1/10 of the 550-nm absorption peak found in a wild-type
strain. The absorption maximum of the remaining ascorbate-reducible
c-type cyt was shifted to 552 nm, consistent with it being
isocyt c2 (33). Moreover,
supernatants of the cyt bc1 mutant
BC17 contained about threefold more cyt c2 than
those of the wild-type strain Ga. The presence of increased amounts of
cyt c2 in supernatants of mutants lacking the
cyt bc1 complex has been observed previously in
R. capsulatus (32).
Cyt c profile of mutants lacking components of the
c-type cytochrome-dependent respiratory branch of R. sphaeroides.
Chromatophore membrane proteins of the
mutants were further analyzed using TMBZ-SDS-PAGE to determine the
profiles of their c-type cytochromes. At least five major
TMBZ-stained bands of molecular masses ranging from approximately 30 to
12 kDa were discernible in the wild-type strain Ga (Fig.
4). Under the separation conditions used
here, the cyt cp subunit of the
cbb3-Cox and cyt c1 subunit of the cyt bc1
complex run together as an unresolved doublet, which was followed by
the cyt co subunit of the
cbb3-Cox and cyt
cy. Cyt c2 ran ahead of
the other c-type cytochromes, but not being membrane
anchored, its amount varied in different samples. Additional
c-type cytochromes of unidentified nature running behind cyt
cp were also visible, and one of them is likely to correspond to the dimethyl sulfoxide-inducible dorC
product (25, 38). As expected, cyt
cy was absent in Gadcy, Gadc2cy, KD03, and KD04;
similarly, Gadc2, Gadc2cy, KD02, and KD05 (Table 1) lacked cyt
c2 (18, 27). Equally, cyt
co was missing in ME127
(cbb3-Cox
aa3-Cox) and in MT001
(cbb3-Cox) and its
derivatives. On the other hand, JS100
(aa3-Cox) was like the
wild-type strain Ga with respect to its c-type cytochrome
content. The overall spectral and TMBZ-SDS-PAGE data established that
the cyt c profiles of various mutants were in agreement with
their genotypes.
View larger version (50K):
[in this window]
[in a new window]
|
FIG. 4.
c-type cytochromes detected in various
R. sphaeroides mutants. Membrane fragments of various
mutants were analyzed by SDS-PAGE, and c-type cytochromes
were revealed via their peroxidase activities using TMBZ as described
in Materials and Methods.
c1+cp,
co, cy, and
c2 correspond to the cyt
c1 and cp subunits of the
cyt bc1 complex and the
cbb3-Cox, cyt
co subunit of the
cbb3-Cox, membrane-attached electron
carrier cyt cy, and soluble electron carrier cyt
c2, respectively. Additional unidentified
c-type cytochromes running behind the doublet corresponding
to cyt cp+c1 are also
visible.
|
|
Respiratory electron flow and Cox activities in
various R. sphaeroides mutants.
Using
the above-described mutant, we probed first for the presence of the
Cox activity in chromatophore membranes of various mutants,
using reduced horse heart cyt c as a substrate. As expected, in semiaerobically grown cells of all strains except the
cbb3-Cox
aa3-Cox mutant ME127,
substantial amounts of Cox activity were detected, although
the cbb3-Cox mutants
exhibited lower levels (data not shown). Next, a detailed characterization of the electron flow through the different components of the respiratory chain was undertaken by using specific substrates and inhibitors. It is known that while addition of NADH or succinate activates electron transport via the respiratory dehydrogenases, inhibitors like antimycin A (Ant A) or Myx block it at the level of the
cyt bc1 complex (Fig. 1) (46).
Respiratory capabilities of various mutants studied here were assessed
directly by monitoring the rates of O2 consumption
depending on NADH or succinate, and in the presence or absence of
exogenous cyt c, as performed previously with various
R. capsulatus mutants (15).
Membrane fragments of the wild-type strain Ga rapidly oxidized various
substrates, and the ensuing O2 uptake was sensitive to Myx,
dropping from 20.6 to 5.8 µmol of O2 consumed/mg of
protein/min in the presence of 2 µM of Myx (Table
3). Addition of 50 µM horse heart cyt
c stimulated the rate of NADH oxidation to 30.9 µmol of
O2 consumed/mg of protein/min. This suggested that
exogenous cyt c substituted well for the shortage of
endogenous cyt c2, which could have been
depleted during cell disruption and membrane fragments preparation.
Exogenous cyt c also greatly increased the rate of NADH
oxidation (from 0.9 to 2.6 µmol of O2 consumed/mg of
protein/min) or ascorbate-Cox activity (from 6.6 to 23.4 µmol of O2 consumed/mg of protein/min) in the cyt
c2 cyt
cy (Gadc2cy), cyt
cy
aa3-Cox (KD03), and
cyt cy
cbb3-Cox (KD04)
mutants lacking the membrane-attached cyt cy
(Table 3). Moreover, pairwise comparison of various strains containing
or lacking cyt cy (i.e., Gadc2 versus Gadcy,
KD02 versus KD04, and KD05 versus KD03) indicated that the presence of
cyt cy led almost in all cases to higher
O2 consumption activities. Thus, cyt
cy plays an important role as an electron
carrier in the respiratory electron transport of R. sphaeroides, for in its absence the respiratory activities
become more dependent on exogenous cyt c.
Remarkably, the artificial electron donor DCIP reduced cyt
cy better than cyt c2, as
indicated by the lower oxidative rates observed in its presence in
appropriate mutants (e.g., cyt cy
cbb3-Cox [KD04]
versus cyt c2
cbb3-Cox [KD02]
mutants [3.6 versus 19.3 µmol of O2 consumed/mg of
protein/min], or cyt cy
aa3-Cox [KD03]
versus cyt c2
aa3-Cox [KD05]
mutants [23.0 versus 88.0 µmol of O2 consumed/mg of
protein/min]) (Table 3). The small amounts of Cox
activities detected in membranes of the cyt
cy
cbb3-Cox (KD04) and
cyt c2 cyt
cy (Gadc2cy) mutants using reduced
DCIP (3.6 and 6.6 µmol of O2 consumed/mg of protein/min,
respectively) also differed from the increase of activity seen upon
addition of cyt c (43.0 and 23.4 µmol of O2
consumed/mg of protein/min, respectively). Thus, DCIP did not act as a
good electron donor when cyt cy and
cbb3-Cox (KD04) or both cyt
c2 and cyt cy (Gadc2cy)
were absent, whereas horse heart cyt c acted as a good
substitute for cyt c2.
Interestingly, no more than one-fifth of the total O2
consumption activity remained upon addition of either 2 µM Myx or 50 µM CN to membrane fragments of the wild-type strain Ga
(Table 3). This remnant activity was in line with NADH oxidation
detected in membranes of the
cbb3-Cox
aa3-Cox (ME127), cyt
c2 cyt
cy (Gadc2cy), or cyt
bc1 (BC17) mutant. Virtually no
Cox was detected in ME127 under all assay conditions,
monitoring either NADH-, succinate-, or ascorbate-dependent O2 consumption, and both in the presence and in
the absence of exogenous cyt c (Table 3). The limited
O2 consumption activity detected was not inhibited by 0.1 mM CN and possibly corresponded to other some kind of
terminal oxidase, such as Qox (13). On the
other hand, the cyt bc1 mutant
BC17 contained Cox activities (39 or 42 µmol of 0.1 mM CN-sensitive O2 consumed/mg of protein/min
when assayed with ascorbate plus DCIP or exogenous cyt c,
respectively) comparable to those seen with the wild-type strain Ga (56 or 57 µmol of O2/mg of protein/min using DCIP or cyt
c, respectively). Lastly, the
cbb3-Cox (MT001) and
aa3-Cox (JS100)
mutants contained about one-half of the total Cox activity found in the wild-type strain Ga (11.7 and 11.1 µmol of
O2/mg of protein/min of NADH oxidase activity in the
presence of exogenous cyt c in JS100 and MT001,
respectively). Clearly, elimination of either the
cbb3-Cox or the
aa3-Cox decreased the total amount of the Cox activity but did not abolish it completely
(37).
Most striking findings were obtained using the cyt
c2
cbb3-Cox (KD02),
cyt cy
aa3-Cox (KD03), cyt
cy
cbb3-Cox (KD04),
and cyt c2
aa3-Cox (KD05)
mutants, which contained simpler electron transport pathways beyond the
cyt bc1 complex (Fig. 1). The data indicated
that higher O2 consumption activities were found in mutants
containing the cbb3-Cox (KD03 and
KD05), and among them that harboring only cyt cy
(i.e., KD05) exhibited the highest Cox activity.
Conversely, lower O2 consumption abilities were encountered
in mutants lacking the cbb3-Cox
(KD02 and KD04), and among them that lacking cyt cy (i.e., KD04) exhibited the lowest activity
(Table 3). Therefore, both cyt c2 and cyt
cy donated electrons to both the
cbb3-Cox and the
aa3-Cox in vitro, but to different
extents. The data suggested that the respiratory electron transfer
pathways via the cbb3-Cox may be
more active than those via the
aa3-Cox subbranches in cells grown
under semiaerobic conditions. However, the amounts of various components varied between various mutants (Table 2), precluding firmer
conclusions. Further dissection of the Cox activities into fractions corresponding to the
cbb3-Cox and
aa3-Cox activities exclusively
linked to cyt c2 or cyt
cy was impossible due to the assays used being
unable to discriminate between the enzymes and their substrates.
NADH-induced cyt c oxidoreduction kinetics in
various R. sphaeroides strains.
NADH-induced
cyt c oxidoreduction kinetics in membranes from
wild-type and various R. sphaeroides mutants were
monitored in the presence or absence of oxygen in order to assess
directly the electron transfer abilities of cyt
cy and cyt c2 to the
cbb3-Cox and
aa3-Cox enzymes. In the wild-type
strain Ga, approximately 50% of the total c-type
cytochromes in membranes were rapidly reduced (half-life
[t1/2] of <1 s) upon addition of
NADH (Fig. 5). This respiration-dependent
steady-state cyt c reduction (i.e., first reduction phase)
lasted for several minutes, depending on the amount of membrane
fragments used, and was sensitive to the cyt bc1
complex inhibitors Myx and Ant A (2 µM each) (Fig. 5). Thus, a large
fraction of the c-type cytochrome complement of membranes
from wild-type cells is in rapid equilibrium with the respiratory
electron transport system. Similar but slower oxidoreduction kinetics (t1/2 = 1 to 2 s) were
observed using membranes from the
aa3-Cox (JS100), cyt
c2
aa3-Cox (KD05), and
cyt cy
aa3-Cox (KD03)
mutants. Even slower cyt c reduction kinetics (2 s,
<t1/2 <10 s) were seen with membranes from the
cbb3-Cox (MT001), cyt
c2
cbb3-Cox (KD02), and
cyt cy
cbb3-Cox (KD04)
mutants.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 5.
Cyt c reduction kinetics in R. sphaeroides wild-type strain Ga and various mutants. Cyt
c reduction kinetics were monitored at 551 to 540 nm using
membrane fragments of various mutants as described in Materials and
Methods. In all experiments, the first reduction wave was initiated by
addition of 0.2 mM NADH at the time indicated by the arrow, while the
second reduction wave followed the onset of anaerobiosis the time of
which was dependent on the rate of oxygen consumption. Dashed traces
represent cyt c reduction patterns observed upon addition of
Ant A or Myx (2 µM each) before the addition of NADH. Protein
concentrations of membrane fragments used were 0.8 mg/ml and 2 to 3 mg/ml for the wild-type strain Ga and its mutant derivatives,
respectively. Below the traces, schemes depicting the available
electron transport pathways are shown.
|
|
Upon depletion of O2, and hence inhibition of the
Cox activities, cyt c reduction further
increased in the wild-type strain Ga and in various
aa3-Cox mutants to
about 95% (second reduction phase). In the
cbb3-Cox mutants, the
amounts of the c-type cytochromes reduced under steady-state
respiration were between 30 and 45% of their total content, reaching a
maximum of about 50 to 60% under anaerobic conditions (Fig. 5). This
second phase of cyt c reduction revealed the amount of cyt
c that had been oxidized during the first phase when cyt
c oxidases were active and hence illustrated at least partly
electron conduction to these enzymes by their electron carriers.
Therefore, the data obtained with mutants containing linear electron
transport pathways between the cyt bc1 complex and the cyt c oxidases demonstrated that both cyt
c2 and cyt cy indeed
donated electrons to either the
cbb3-Cox or
aa3-Cox enzyme. However, the cyt
cy aa3-Cox (KD02) and cyt
c2 aa3-Cox (KD04) pathways appeared to
be less efficient (i.e., smaller second phases), suggesting that under
our experimental conditions the
aa3-Cox played in O2
consumption a less prominent role than the
cbb3-Cox.
The light-harvesting antenna complexes are induced in the presence of
oxygen in R. sphaeroides mutants lacking either both cyt
c2 and cyt cy or the
cbb3-Cox activity (28,
29). During our studies we also noted that R. sphaeroides strain MT001 lacking the
cbb3-Cox and its derivatives lacking
in addition either cyt c2 or cyt
cy (KD02 or KD04) formed highly pigmented
colonies under Res growth conditions. This pigmentation was especially
pronounced in a mutant lacking both cyt cy and
the cbb3-Cox (KD04). Indeed, spectral examination of chromatophore membranes of various mutants confirmed the presence of prominent absorption peaks in the 800- to
875-nm region (Fig. 6). Thus, the absence
of the cbb3-Cox and its electron
donors modulated the production of the light-harvesting complexes in
the presence of oxygen. Remarkably, the pigment complexes were either
absent or much less pronounced in the other R. sphaeroides Ga derivaties used in this work, including the
cbb3-Cox
aa3-Cox (ME127), cyt
bc1 (BC17), and cyt
c2 cyt
cy (Gadc2cy) mutants.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 6.
Absorption spectra of membrane fragments of various
R. sphaeroides mutants. Membrane fragments were prepared
from cells grown by respiration at an optical density (OD) at 630 nm of
0.4, and their absorption spectra were recorded as described in
Materials and Methods. Spectra were obtained for the wild-type strain
Ga and the cyt c2 (Gadc2), cyt
cy (Gacy), cyt
c2 cyt
cy (Gadc2cy),
cbb3-Cox
aa3-Cox (ME127), and
cyt bc1 (BC17) mutants (A), the
wild-type strain Ga and the
cbb3-Cox (MT001), cyt
c2
cbb3-Cox (KD02), and
cyt cy
cbb3-Cox (KD04)
mutants (B), and the wild-type strain Ga and the
aa3-Cox (JS100), cyt
cy
aa3-Cox (KD03), and
cyt c2
aa3-Cox (KD05) mutants
(C). See the text for further details.
|
|
| |
DISCUSSION |
In this work we used a biochemical genetic approach, which
consisted of disrupting simultaneously one of the two electron carrier
c-type cytochromes and one of the two cyt c
oxidases, to dissect the complicated respiratory electron transport
pathways of R. sphaeroides (Fig. 1). The double mutants
thus obtained simplified the crisscrossed pathways and enabled us to
analyze the abilities of the different electron carriers to convey
electrons from the cyt bc1 complex to the
terminal cyt c oxidases. Prior to this work, it was well
known that the soluble cyt c2 donates electrons to the aa3-Cox; indeed, it could be
assumed correctly that it also donates electrons to the
cbb3-Cox (15, 27). On
the other hand, not much was known about the electron carrier
properties of the more recently discovered membrane-anchored cyt
cy in R. sphaeroides (27,
51). Studies reported here confirmed for cyt
c2, and demonstrated for the first time for cyt
cy, that both are efficient electron donors to
both the cbb3-Cox and the
aa3-Cox during the respiratory
growth of R. sphaeroides. Neither of the two electron
carriers is restricted to function solely with either of the cyt
c oxidases, and clearly all four subbranches appear to be
functional (Fig. 1). Whether or not all of these pathways are also
proficient in vivo to support respiratory growth of R. sphaeroides in the absence of its Qox-dependent
alternate branch(es) remains to be seen.
At least under the semiaerobic growth conditions used here, the
cbb3-Cox subbranches appear to be
more prominent in catalyzing electron transfer between the cyt
bc1 complex and the cyt c oxidases in
vitro. However, whether this prominence is due to the larger amounts of
the components of these pathways or to their better catalytic abilities
remains unknown until determination of their amounts in each case.
Among the electron carriers, while cyt c2 can diffuse between the RC and the cyt bc1
complexes of the photosynthetic electron transfer chains
(10), cyt cy is conceivably
restricted to interact with a limited number of partners due to its
membrane anchor (26, 27). Nonetheless, it is noteworthy
that the alpha group proteobacterium Rickettsia prowazekii,
thought to be closely related to mitochondria, lacks a soluble cyt
c but contains a membrane-attached cyt
cy homologue (1, 23).
Availability of otherwise isogenic mutants lacking different
c-type cytochromes of R. sphaeroides allowed
us to identify each of these proteins unambiguously and confirmed our
previous assignments of the cytochromes c2,
cy, c1,
co, and cp in
R. sphaeroides (18, 27). It is
noteworthy that in this bacterium cyt cy runs
ahead of the cyt co subunit of the
cbb3-Cox (Fig. 3), which is
different from what has been observed with R. capsulatus cyt
cy (26). Whether the inability of
R. sphaeroides cyt cy to donate
electrons to the RC is linked to its smaller size remains to be
seen (27).
The mutants described here, eliminating systematically various
respiratory components, allowed us to estimate the amounts of
various b- and c-type cytochromes present in
membranes of R. sphaeroides cells grown under
semiaerobic conditions. It appears that in a wild-type strain like Ga,
elimination of the cbb3-Cox decreased roughly one-half of the total amount of the b-type
cytochromes and two-thirds of the c-type cytochromes in
membranes. Similarly, deletion of the cyt bc1
complex also decreased about one-half of the total amount of both
b- and c-type cytochromes. These estimations are
in agreement with the subunit composition of the
cbb3-Cox and the cyt
bc1 complex and suggest that these complexes may
be present at similar amounts in R. sphaeroides
membranes under the growth conditions used here. However, it should be
noted that the amounts of various cytochromes change in different
mutants as a function of the presence or absence of various components of the electron transport chain. For example, the absence of
electron-carrying c-type cytochromes or the cyt
bc1 complex also decreased the amounts of the
cbb3-Cox or the
aa3-Cox (Table 3). Thus, caution
should be exercised in extrapolating the results obtained using
membrane fragments to intact cells grown under various physiological conditions.
Estimation of the Cox-independent respiratory pathway in
the wild-type strain Ga and in
cbb3-Cox
aa3-Cox (ME127), cyt
bc1 (BC17), and cyt
c2 cyt
cy (Gadc2cy) mutants indicated
that in R. sphaeroides about one-fifth of the
respiratory capacity is insensitive to 100 µM CN
and is possibly mediated via a Qox enzyme(s).
This situation is unlike that observed in R. capsulatus
(15), where the analogous CN-resistant NADH-dependent
respiration represents about 75 to 85% of NADH oxidation rate.
Moreover, in R. sphaeroides, restraining the
mitochondrial-like respiratory electron flow by eliminating some of its
components does not affect the levels of CN-resistant respiration. Membranes of an R. sphaeroides mutant
lacking the cyt c2 and cyt
cy (Gadc2cy) exhibit very low NADH oxidation
activity (Table 3), unlike its R. capsulatus counterpart FJ2
(cyt c2 cyt
cy), which has a CN-resistant NADH
oxidation activity roughly three times higher than that of its parental
wild-type strain MT1131 (15). This phenomenon was
suggested to reflect a regulatory response of one of the
respiratory branches over the other (49), but the
molecular basis of this effect is unknown. No similar response is
obvious in R. sphaeroides, for the mutants analyzed here
exhibited a CN-resistant respiratory activity similar to that present
in the wild-type membranes. The marked inefficiency of the alternate
respiratory pathway, in addition to the photosynthetic incompetence of
cyt cy and the presence of an
aa3-Cox, constitutes yet another
striking difference between the growth abilities of the closely related
species R. capsulatus and R. sphaeroides.
Increased synthesis of the photosynthetic apparatus in the presence of
oxygen in mutants lacking the
cbb3-Cox has been documented previously in R. sphaeroides strain 2.4.1 (28,
52). Recently, a similar situation has also been described in
derivatives of the same strain lacking either the cyt
bc1 complex or the cytochromes c2 and cy
(29). In these mutants, expression of the photosynthetic apparatus is not repressed by O2, unlike in the wild-type
strain, thus leading to the accumulation of increased amounts of
light-harvesting antenna complexes and photosynthetic pigments. In
R. sphaeroides strain Ga and its derivatives used here,
the light-harvesting complexes were also highly induced in the
cbb3-Cox (MT001), cyt
c2
cbb3-Cox (KD02), and
especially cyt cy
cbb3-Cox (KD04)
mutants, while this induction was less apparent in the cyt
bc1 (BC17),
cbb3-Cox
aa3-Cox (ME127), and
cyt c2, cyt
cy (Gadc2cy) mutants. Why similar
mutants derived from R. sphaeroides strains 2.4.1 and Ga
behave differently in this respect is unclear. In any event, close
examination of the electron transport pathways (Fig. 1) suggests that
either restricting the electron flow via the
cbb3-Cox subbranches, as proposed
earlier (29, 52), or forcing this flow via the
aa3-Cox subbranches, or even a
combination of these possibilities, may lead to the accumulation of the
pigment proteins. Clearly, the creation of an imbalance between the
aa3-Cox and
cbb3-Cox subbranches without taking
into account the availability of O2 might explain at least
partly the pigmentation phenotypes observed in various mutants used in
this work.
In summary, the work presented here establishes for the first time that
both of the electron carriers cyt c2 and cyt
cy are efficient electron donors to both the
cbb3-Cox and the
aa3-Cox during the respiratory
growth of R. sphaeroides. It remains to be seen whether
or not the electron flow via each of the subbranches defined here is
alone sufficient to support the Res growth of R. sphaeroides under physiological growth conditions.
This work was supported by DOE grants 91ER20052 to F.D.,
CRP/TUR99-01 to S.M., and MURST-COFIN 99 to D.Z.
| 1.
|
Andersson, S. G.,
A. Zomorodipour,
J. O. Andersson,
T. Sicheritz-Ponten,
U. C. M. Alsmark,
R. M. Podowski,
A. K. Naslund,
A.-S. Ericksonn,
H. H. Winkler, and C. G. Kurland.
1998.
The genome sequence of Rickettsia prowazekii and the origin of mitochondria.
Nature
396:133-140[CrossRef][Medline].
|
| 2.
|
Bradford, M. M.
1976.
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[CrossRef][Medline].
|
| 3.
|
Caffrey, M.,
E. Davidson,
M. Cusanovich, and F. Daldal.
1992.
Cytochrome c2 mutants of Rhodobacter capsulatus.
Arch. Biochem. Biophys.
292:419-426[CrossRef][Medline].
|
| 4.
|
Choudhary, M.,
C. Mackenzie,
N. J. Mouncey, and S. Kaplan.
1999.
RsGDB, the Rhodobacter sphaeroides genome database.
Nucleic Acids Res.
27:61-62[Abstract/Free Full Text].
|
| 5.
|
Clayton, R. K.
1963.
Spectroscopic analyses of bacteriochlorophylls in vitro and in vivo.
Photochem. Photobiol.
5:669-677.
|
| 6.
|
Daldal, F.,
E. Davidson, and S. Cheng.
1987.
Isolation of the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1, all components of the ubiquinol: cytochrome c oxidoreductase complex of Rhodopseudomonas capsulata.
J. Mol. Biol.
195:1-12[CrossRef][Medline].
|
| 7.
|
Daldal, F.,
S. Cheng,
J. Applebaum,
E. Davidson, and R. C. Prince.
1986.
Cytochrome c2 is not essential for photosynthetic growth of Rhodopseudomonas capsulata.
Proc. Natl. Acad. Sci. USA
83:2012-2016[Abstract/Free Full Text].
|
| 8.
|
Donohue, T. J.,
A. G. McEwan,
S. Van Doren,
A. R. Crofts, and S. Kaplan.
1988.
Phenotypic and genetic characterization of cytochrome c2 deficient mutants of Rhodobacter sphaeroides.
Biochemistry
27:1918-1925[CrossRef][Medline].
|
| 9.
|
Fellay, R.,
J. Frey, and H. Krisch.
1987.
Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria.
Gene
52:147-153[CrossRef][Medline].
|
| 10.
|
Fernandez-Valesco, J., and A. R. Crofts.
1991.
Complexes or supercomplexes: inhibitor titrations show that electron transfer in chromatophores from Rhodobacter sphaeroides involves a dimeric UQH2:cyt c2 oxidoreductase, and is delocalized.
Biochem. Soc. Trans.
19:588-593[Medline].
|
| 11.
|
Fonstein, M.,
T. Nikolskaya,
Y. Kogan, and R. Haselkorn.
1998.
Genome encyclopedias and their use for comparative analysis of Rhodobacter capsulatus strains.
Electrophoresis
19:469-477[CrossRef][Medline].
|
| 12.
|
Garcia-Horsman, J. A.,
E. Berry,
J. P. Shapleigh,
J. O. Alben, and R. B. Gennis.
1994.
A novel cytochrome c oxidase from Rhodobacter sphaeroides that lacks CuA.
Biochemistry
33:3113-3119[CrossRef][Medline].
|
| 13.
|
Garcia-Horsman, J. A.,
B. Barquera,
J. Rumbley,
J. Ma, and R. B. Gennis.
1994.
The superfamily of heme-copper respiratory oxidases.
J. Bacteriol.
176:5587-6000[Free Full Text].
|
| 14.
|
Gray, K. A.,
M. Grooms,
H. Myllykallio,
C. Moomaw,
C. Slaughter, and F. Daldal.
1994.
Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center.
Biochemistry
33:3120-3127[CrossRef][Medline].
|
| 15.
|
Hochkoeppler, A.,
F. E. Jenney, Jr.,
S. E. Lang,
D. Zannoni, and F. Daldal.
1995.
Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration.
J. Bacteriol.
177:608-613[Abstract/Free Full Text].
|
| 16.
|
Hosler, J. P.,
J. Fetter,
M. M. Tecklenburg,
M. Espe,
C. Lerma, and S. Ferguson-Miller.
1992.
Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping and spectral properties.
J. Biol. Chem.
267:24264-24272[Abstract/Free Full Text].
|
| 17.
|
Jenney, F. E., and F. Daldal.
1993.
A novel membrane-associated c-type cytochrome, cyt cy can mediate the photosynthetic growth of Rhodobacter capsulatus and Rhodobacter sphaeroides.
EMBO J.
12:1283-1292[Medline].
|
| 18.
|
Jenney, F. E.,
R. C. Prince, and F. Daldal.
1996.
The membrane-bound cytochrome cy of Rhodobacter capsulatus is an electron donor to the photosynthetic reaction of Rhodobacter sphaeroides.
Biochim. Biophys Acta
1273:159-164[Medline].
|
| 19.
|
Koch, H.-G.,
O. Hwang, and F. Daldal.
1998.
Isolation and characterization of Rhodobacter capsulatus mutants affected in cytochrome cbb3 oxidase activity.
J. Bacteriol.
180:969-978[Abstract/Free Full Text].
|
| 20.
|
Koch, H.-G.,
C. Winterstein,
A. S. Saribas,
J. O. Alben, and F. Daldal.
2000.
Roles of the ccoGHIS gene products in the biogenesis of the cbb3-type cytochrome c oxidase.
J. Mol. Biol.
297:49-65[CrossRef][Medline].
|
| 21.
|
La Monica, R. F., and B. L. Marrs.
1976.
The branched respiratory system of photosynthetically grown Rhodopseudomonas capsulata.
Biochim. Biophys. Acta
423:431-439 |
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
Present address: Institut de Génétique et
Microbiologie, Université Paris XI, 91405 Orsay Cedex, France.
