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INTRODUCTION |
Hydrogenase catalyzes the reversible
reaction H2
2H+ + 2e
(EC
1.18.99.1 and 1.12.2.1) (12). The enzyme occurs in a wide variety of eubacteria and archaea, and the catalytic direction depends
on cellular location and metabolic function. Azotobacter vinelandii has a membrane-bound hydrogenase that primarily
catalyzes the oxidative reaction (38). Membrane-bound
[NiFe] hydrogenases consist of a membrane-bound catalytic heterodimer
and an integral membrane cytochrome b (12). While
the heterodimer alone can catalyze hydrogenase reactions with
artificial electron acceptors and donors, the cytochrome b
is essential for physiologically relevant H2 oxidation
(8-10, 20, 26, 40). Proposed functions for HoxZ, the
cytochrome b in A. vinelandii, include electron transfer from the heterodimer to the respiratory chain, anchoring the
heterodimer to the membrane, reduction of the catalytic site, and
stabilization of the enzyme (40).
All three subunits of membrane-bound hydrogenases are metalloproteins,
and the ligands in the subunits have unusual structural and functional
features. The large subunit contains the site of catalysis, and the
iron in the [NiFe] catalytic site has the nonprotein ligands CO and
CN (18). The small subunit of the heterodimer
has three aligned FeS clusters (45). Irons in FeS clusters
are usually ligated by cysteines, but the distal 4Fe4S cluster includes
a histidine ligand (45), which is essential for activity
(16). Substitution of one of the cysteine ligands of the
central 3Fe4S cluster in A. vinelandii resulted in the loss
of O2 sensitivity in hydrogenase, which is normally highly
sensitive to O2 inactivation (25). The hemes and
ligands of the third subunit have been the subject of recent studies
(5, 7, 9, 16).
Dross et al. (9) determined that the integral membrane
component of hydrogenase in Wolinella succinogenes, HydC,
had four hydrophobic segments, and they suggested that this protein was a cytochrome b. From their alignment of full and partial
homologous sequences to HydC, they indicated proposed 
-helical
transmembrane segments and two heme ligands. However, our alignment of
these hydrogenase b-type cytochromes, which included
subsequently released full sequences of Azotobacter
chroococcum (10) and Bradyrhizobium japonicum (44), suggested that four conserved
histidines may serve as ligands to two hemes, in agreement with Berks
et al. (7) and Gross et al. (16). The homologous
protein in Alcaligenes eutrophus, HoxZ, was shown to be a
diheme cytochrome b that anchors the heterodimer to the
periplasmic side of the cytoplasmic membrane and donates electrons to
ubiquinone (6). When three of the conserved histidines in
HydC of W. succinogenes were replaced with alanine or
methionine, the mutant strains had negligible levels of
H2-dependent electron transfer to the anaerobic electron acceptor, menaquinone (16). The fourth conserved histidine
was suggested as the fourth ligand (7, 16) but was not
identified experimentally. The requirement of three ligands for
physiologically relevant electron transfer from H2 was
shown (16), but further investigations of the roles of these
ligands have not been carried out.
Unlike W. succinogenes, A. vinelandii is an
aerobic, nitrogen-fixing bacterium that has ubiquinone as part of the
respiratory chain. Along with the four conserved histidines, there are
five histidines conserved to various degrees among 14 hydrogenase
b-type cytochrome homologues. Four of these five residues
occur in A. vinelandii HoxZ but not in W. succinogenes. We have investigated the effect of histidine
substitutions in both O2-dependent H2 oxidation
and H2-dependent methylene blue (MB) reduction in vivo. Substitution of histidine residues with nonligands (alanine), alternative ligands (tyrosine), and exogenous ligands (Imd) provided a
means of identifying the heme ligands and probing the heme site and the
role of the ligands in HoxZ. Imd rescued mutants with substitutions in
one of the putative heme ligands. This is the first report of imidazole
(Imd) rescue of activity in a membrane protein and the first report of
in vivo rescue in a nonheterologous system.
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MATERIALS AND METHODS |
Schematic of A. vinelandii HoxZ.
The sequence of
A. vinelandii HoxZ (National Center for Biotechnology
Information [NCBI] GenBank accession no. L23970, submitted by Menon
et al. [27]) is shown in Fig.
1. The designated transmembrane segments
were most similar in selected residues and length to those predicted by
NCBI Entrez protein query for accession no. P23000 (A. vinelandii HoxZ).
Plasmids, bacterial strains, and mutagenesis.
The plasmids
and bacterial strains used in this study are listed in Table
1. Oligonucleotides for the mutagenesis
were designed using the software Oligo (version 3; Molecular Biology
Insights, Plymouth, Minn.). The oligonucleotide-directed PCR-based
methods of Merino et al. (29) and Weiner et al.
(46) were used for site-directed mutagenesis of the plasmid
template pAVHoxZ+. The PCR product was then transformed
into competent Escherichia coli DH5 (Gibco BRL, Grand
Island, N.Y.), which were plated on Luria-Bertani agar medium
(39) supplemented with ampicillin (60 mg/liter) (Sigma, St.
Louis, Mo.) and grown at 37°C overnight. Single colonies were grown
overnight in ampicillin-supplemented Luria-Bertani liquid medium, and
plasmid preparations were made according to Lee and Rasheed
(21). Sequencing by the Central Services Lab of the Center
for Gene Research and Biotechnology, Oregon State University, was used
to screen and verify the mutants.
Growth of A. vinelandii.
A. vinelandii strains
were grown at 30°C in Burk medium or on Burk plates (43).
For rifampin-resistant (Rifr) strains, the medium was
supplemented with 20 mg of rifampin (Sigma) per liter (making Burk-Rif
medium). Cultures were shaken continuously (150 cycles/min) during
growth and grown to optical densities at 600 nm (OD600) of
0.8 to 1.1 with a Beckman DU-70 spectrophotometer, except for the
wild-type (Wt) activity assay, in which case cultures were grown to
OD600 of 0.5 to 1.2. For Imd (Sigma)-grown cultures, Imd
(made to pH 7.5 with HCl) was added to a final concentration of 10 mM
(4) at the time of inoculum addition. Cells were collected
by centrifugation.
Transformation and screening.
A. vinelandii strains
were made competent by several transfers on iron- and
molybdenum-deficient, 28 mM ammonium acetate-supplemented Burk medium
(36) under O2-limited conditions
(33). To generate a Rifs HoxZ
A. vinelandii strain, pHoxZ (1 µg) was
transformed into competent A. vinelandii DJ (Wt) without the
aid of an antibiotic marker. Except for growth in Burk medium without
rifampin, other aspects of transformation and screening were the same
as for other mutants. Transformations with all other HoxZ constructs
were done using plasmids linearized with the restriction enzyme
SmaI (U.S. Biochemical, Cleveland, Ohio; Promega, Madison, Wis.). Linearized plasmid preparations (1 µg of DNA) of pHoxZ H33,
H74, H194, H208, and H194,208
A or -Y were individually cotransformed with the EcoRI (Promega) 1.7-kb fragment of pDB303 (10 ng),
which confers Rifr into Rifs A. vinelandii DJ (Wt), using the method of Premakumar et al. (36). Linearized plasmids pHoxZ H97, H98, H136, and H191A
or -Y were cotransformed similarly into Rifs A. vinelandii HoxZ. To compare only hox
mutants and not differences due to the lower growth rate in medium with
rifampin, A. vinelandii DJ and A. vinelandii HoxKG were made Rifr by transformation using
the 1.7-kb EcoRI fragment of pDB303. The day following
transformation, bacteria were transferred to Burk-Rif agar plates to
select for Rifr transformants. Single colonies were picked
and transferred three times on Burk-Rif medium to allow for complete
segregation (22, 36). A phenotypic assay of H2
oxidation ability similar to that of Sayavedra-Soto and Arp
(40) was used to screen for coincident transformation of the
HoxZ mutants. Test tubes containing 2 ml of Burk-Rif were inoculated
from single colonies; cultures were grown to an OD600 of
0.4 and capped with butyl rubber stoppers, and H2 (4.46 µmol) was added (high-purity commercial grade H2 and
N2 were purchased locally). Cultures were then grown on a shaker table (150 rpm) for 16 h at 30°C. For screening, culture headspace was sampled and analyzed for the presence of H2
using a Shimadzu GC-8A thermal conductivity detector gas chromatograph (Molecular Sieve 5A column; 5 ft long, 1/8-inch diameter, 200°C injection temperature, 120°C column temperature, 60-mA current). Strains with phenotypes differing from that of the background parental
strain were confirmed as mutants by DNA sequence analysis of a PCR
product of the genomic DNA preparation (3). Sequencing was
performed at the Central Services Lab of the Center for Gene Research
and Biotechnology, Oregon State University.
Whole cell O2-dependent H2
oxidation.
To determine when to harvest A. vinelandii
for assays, a Wt activity assay compared OD600 (0.5 to 1.2)
and protein contents (microbiuret assay [14] with
bovine serum albumin as a standard) to O2-dependent
H2 oxidation activity. A. vinelandii cultures for other assays were then harvested in the range of greatest activity,
OD600 of 0.8 through 1.0. The specific activities in nanomoles of H2 oxidized per minute per milligram at these
ODs were 0.8 (188), 0.9 (184), and 1.0 (173). Preliminary experiments showed that cells resuspended in growth medium had H2
oxidation activity, but cells resuspended in phosphate buffer did not,
probably due to oxidation of the hydrogenase enzyme. Therefore 3 mM
sucrose, equivalent to a starvation level of carbon for E. coli (0.1% glucose) (2), was added to the buffer to
maintain respiratory protection of O2-sensitive enzymes.
Suspension cultures (1.5 ml) were centrifuged for 1 min at 14,000 rpm,
resuspended in 50 mM phosphate buffer (pH 7.0)-3 mM sucrose (1.5 ml),
then placed in 10-ml serum vials, capped with a rubber sleeve stopper,
and placed upside down in a 30°C water bath at a shaker rate of 200 cycles/min. To initiate the assay, H2 (4.46 µmol) was
added to the vial. Headspace samples (200 µl) were injected into the
thermal conductivity detector gas chromatograph at 0, 15, and 30 min.
For assays with Imd, either cultures were grown in 10 mM Imd, or Imd
and chloramphenicol (Aldrich Chemical Company, Milwaukee, Wis.) were
added at the initiation of the experiment to final concentrations of 10 and 3 mM, respectively. Four individual replications of this assay were
recorded for each strain. In all assays, results for the control
strains grown with Imd were not significantly different (least squares
difference at P = 0.05 [LSD0.05]) from
the results for control strains grown without Imd.
Whole cell, H2-dependent MB reduction.
Suspension cultures (40 ml) were centrifuged 10 min at 7,500 rpm; the
cells were resuspended in 1 ml of 50 mM Tris buffer-5 mM EDTA (pH 7.3)
and placed in a 10-ml serum vial stoppered with a rubber sleeve cap.
The vial was purged with N2 to remove O2. A
sample cuvette (7.5 ml; Beckman 2097) containing a total of 1 ml of 50 mM Tris buffer-5 mM EDTA (pH 7.3) including 150 µM MB was similarly
stoppered and flushed with N2, and H2 (1 ml) was added for assays with H2. All reactions were started
with the addition of N2-purged culture (50 µl) to the
sample cuvette. Under the conditions of these MB experiments,
activation was not required for reduction of the catalytic site since
the heterodimers were sufficiently reduced from the flushing of
nitrogen gas and removal of O2 to reduce MB without a lag
period and in the absence of dithionite. The decrease in absorbance at
690 nm was monitored with a Beckman DU-70 spectrophotometer for 1 min,
and the greatest difference in absorbance in a 10-s time period was
recorded. Four individual replications of this assay were recorded for
each strain. Calculations were based on a molar extinction coefficient
of 11.4 mM1 cm1 at 690 nm. MSU Stat
(Montana State University, Bozeman) software was used for analysis of
variance of the means for the data from both O2-dependent
H2 oxidation and H2-dependent MB reduction assays.
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RESULTS |
Conservation of histidines in hydrogenase b-type
cytochromes.
An alignment and analysis of 14 eubacterial
hydrogenase b-type cytochromes (alignment not shown) show
that four conserved histidines are found in the predicted transmembrane
-helical segments (Fig. 1). One histidine is near the center of
helix A, one is at the beginning of helix B, and two are in helix D, in agreement with Gross et al. (16). Six histidines occur in
A. vinelandii without corresponding histidines in W. succinogenes. Four of these in the loop regions were selected for
mutagenesis. Three of the four are in loop BC, predicted to be on the
cytoplasmic side, and the fourth is in loop CD, predicted to be on the
periplasmic side of the cytoplasmic membrane (Fig. 1). A. vinelandii HoxZ is only distantly related (29% identical) to the
hydrogenase cytochrome b of W. succinogenes
(phylogeny not shown). However, the similarities seen in biochemical
assays of these distantly related proteins imply similar structural and
functional importance of all hydrogenase b-type cytochromes.
To examine the roles of these histidines in HoxZ, mutants were
constructed to produce altered proteins with specific histidines
replaced with either alanine or tyrosine.
Effect of histidine replacements on whole cell
O2-dependent H2 oxidation.
To determine
whether hydrogenase is functional in physiological electron transport
in A. vinelandii hoxZ mutants, H2 oxidation with
O2 as the terminal electron acceptor was assayed (Table
2). A. vinelandii DJ (Wt) and
Rifr A. vinelandii DJ (WtR) served as positive
controls. HoxKG Rifr (a partial deletion
mutant of the catalytic heterodimer) served as a negative control.
HoxZ Rifr (a partial deletion mutant of
hydrogenase cytochrome b) served as a control for the loss
of a functional HoxZ. Wt and WtR were not significantly different.
Physiologically relevant H2 oxidation activity was
undetectable for both Hox mutants.
No H2 oxidation was detected for the mutants with alanine
substitutions in putative ligand positions 33, 74, 194, and 208 (Table
2). None of these HisAla mutants, including the mutant, H194,208A
were functional in physiological electron transport from hydrogenase to
O2. These positions are the four conserved histidines in
aligned sequences of hydrogenase b-type cytochromes and are
therefore proposed as the ligands to the two hemes in HoxZ.
Histidines that were not fully conserved were not expected to be
ligands to the hemes. Four of these histidines in loop regions near the
membrane edges were replaced in A. vinelandii by alanine or
tyrosine. H2 oxidation assays of putative nonligand mutants indicated that all four had H2 oxidation activity and that
three of the four mutants (with replacements in positions 97, 98, and 191) had similar to Wt activity (Table 2). Residues 97 and 98 are
predicted to be on the cytoplasmic side of the plasma membrane, and 191 is predicted to be on the periplasmic side. Mutant H136A had
approximately one-third of Wt activity. Residue 136 is predicted to be
on the cytoplasmic side of the membrane, and the decreased ability of
H136A to oxidize H2 implies that the histidine in position 136 serves an important function.
Mutants with HisTyr substitutions in positions 33, 74, and 208 showed that tyrosine in these positions gave the same result as the
HisAla mutants (Table 2). In contrast, mutant H194Y appeared to have
some H2 oxidation activity (consumption in 30 min of ~5% of the initial H2 added, which is equivalent to 23% of Wt
activity) and is significantly different from both Wt and the control
with no cells (Table 2).
Effect of Imd on whole cell O2-dependent H2
oxidation of mutants.
In some proteins, Imd can serve as an
exogenous ligand to heme in proteins where a natural ligand has been
removed (4). To determine if Imd, a histidine analogue,
could serve as a free ligand in physiologically relevant H2
oxidation, 10 mM Imd was added to the growth medium. There was no
significant difference in H2 oxidation between Wt and WtR,
with or without Imd (Table 2). Furthermore, Imd did not restore
activity to HoxKG Rifr or HoxZ
Rifr.
Addition of 10 mM Imd to the growth medium of HisAla ligand mutants
33, 74, and 208 did not increase H2 oxidation activity (Table 2). Surprisingly, H194A recovered activity to a level comparable
to Wt (Table 2; see Fig. 3). Hydrogenase activity in Imd-treated H194A
[H194A (Imd)] indicates that Imd acts as a free ligand in this
position and reconstitutes hydrogenase activity to Wt levels. Over the
range of 1 to 10 mM Imd, H2 oxidation rates by H194A (Imd)
and Wt (Imd) were not significantly different from Wt (data not shown).
When Imd was excluded from the growth medium but added at the start of
the assay, H2 oxidation activity was detected immediately
and with no change throughout the course of the assay (Fig.
2). However, the rate (0.38 nmol/min/ml)
was lower than when cells were grown with Imd (0.88 nmol/min/ml) (Fig. 2) (LSD0.05 = 0.11). Addition of chloramphenicol, a
known inhibitor of protein synthesis in A. vinelandii
(34, 37), did not prevent the restoration of activity by
Imd. These results indicate that new protein synthesis was not required
for Imd to bind and serve as a ligand in trans.
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FIG. 2.
Recovery of O2-dependent H2
oxidation by H194A with the addition of Imd. Symbols represent H194A
alone ( ), H194A plus Imd added at initiation of assay ( ), H194A
plus Imd and chloramphenicol added at initiation of assay ( ), H194A
grown in Imd ( ), and no-cell control ( ).
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The HisTyr ligand mutants grown with Imd followed a similar pattern
as the HisAla ligand mutants in that only H194Y had activity when
grown in Imd (Table 2). A comparison of both mutants grown in Imd shows
that there is no significant difference among Wt, H194A (Imd), and
H194Y (Imd) (LSD0.05 = 0.13) (additional data not shown).
The nonligand HoxZ mutants were also grown with Imd and assayed for
O2-dependent H2 oxidation. Activities of the
nonligand mutants grown with Imd were similar to activities of these
mutants grown without Imd. H136A did not recover
H2-oxidizing capability upon addition of Imd. We assumed
that HoxZ was expressed to Wt levels in all cases since the nonligand
mutants and one ligand mutant grown with Imd have high levels of
activity. Because antibody to HoxZ was not available, expression of the
altered proteins in HoxZ mutants was not assayed for directly.
Whole cell H2-dependent MB reduction.
MB reduction
can be quantitatively measured spectroscopically and must be assayed
anaerobically to prevent O2 from reoxidizing the artificial
electron acceptor. MB can accept electrons directly from the
heterodimer (42) and may have other nonspecific binding locations along the electron transport chain and in the cell. Since MB
can also be nonspecifically reduced, experiments were conducted in the
absence and presence of H2 (1) (Table
3) to account for the level of endogenous
cellular reduction. In addition, assays were conducted on strains grown
in the absence and presence of Imd.
Analysis of the control strains of Wt and the partial deletion mutants
show that without H2, all three strains have similar levels
of endogenous reduction of MB. The endogenous level of Wt MB reduction
was approximately 32% of total Wt MB reduction when H2 was
added. Without H2, all mutant strains also had similar but
low levels of endogenous activity, comparable to HoxKG
with or without H2 (LSD0.05 = 4.8) (Table
3). However, with H2, the controls showed three distinct
levels of activity relative to the endogenous level of MB reduction:
none, partial, and full. H2 did not stimulate reduction of
MB by the KG deletion mutant, which is consistent with no hydrogenase
heterodimer activity. H2 stimulated MB reduction by
HoxZ, indicating a functional hydrogenase heterodimer,
but MB reduction was 70% of the Wt level (partial activity). The lack
of a complete HoxZ affected the ability of the heterodimer to reduce
MB. The H2-dependent MB reduction of Wt was considered to
be full activity. In all strains except HoxKG, when
H2 was added, reduction of MB was also immediate, but more rapid than in the absence of H2, which indicates that the
heterodimers are functional (LSD0.05 = 5.6) (Table 3).
In assays with H2, MB reduction rates by ligand mutants
varied with position (Fig. 3) but were
similar between alanine and tyrosine mutants at the same position
(tyrosine substitution data not shown). H33A/Y had full activity,
regardless of the presence of Imd. Mutants of position 74 had partial
activity, whether Imd was present or not. Positions H194A/Y and H208A/Y
individually had partial activity without Imd and full activity with
Imd. The double ligand mutant H194,208A with Imd showed substantial
increase in activity but did not regain full activity.
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FIG. 3.
Effect of Imd on H2-dependent MB reduction
and O2-dependent H2 oxidation. Bars denote
standard error; only top half of column error is shown. The
x axis indicates A. vinelandii controls and HA
mutants.
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With or without Imd, three of the four nonligand mutants had at least
full Wt activity (Table 3). With H2, but with or without Imd, H136A had significantly greater (>20% increase) MB reduction than Wt and was the only strain with significantly greater than Wt
activity. H97Y was unusual in that it had less than Wt activity when
assayed without Imd but was comparable to Wt in
H2-dependent MB reduction when grown with Imd (Table 3).
The MB reduction results indicate that the heterodimers in the HoxZ
partial deletion mutant and point mutants were catalytically active and
that the lack of activity in the O2-linked H2
oxidation assay was localized to HoxZ. The rates of reduction in
HoxZ and three of the ligand mutants were not comparable
to Wt, which indicates that the heme ligands in HoxZ are required for
full catalytic capability of the hydrogenase heterodimer. Unlike their roles in electron transport, in which each ligand was required for
hydrogenase function, the four ligands do not play equivalent structural roles, as shown by MB reduction (Fig. 3). Depending on the
position, Imd differs in its ability to substitute for ligands and
fulfill their functional or structural role.
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DISCUSSION |
Influence of residue substitutions in HoxZ on
O2-dependent H2 oxidation.
In A. vinelandii HoxZ, the four histidines 33, 74, 194, and 208 are
essential for physiologically relevant hydrogenase activity (Table 2)
and are proposed as the ligands to two hemes in this cytochrome
b. The first three residues are homologous to histidines in
W. succinogenes HydC, which when substituted with other
residues also resulted in cells with low or no physiologically relevant hydrogenase activity (16). In several quinone-interacting
b-type cytochromes (7, 11, 16, 49), four
conserved histidines occur in transmembrane segments. The invariant
histidines in these proteins have been suggested to be ligands to two
hemes. In succinate:ubiquinone oxidoreductase (11) and the
bc1 complex (49), residue
replacements of conserved histidines resulted in loss of activity and
changes in absorption spectra, which supported the conclusion that
these histidines were ligands to two hemes. Crystal structures of
various bc1 complexes confirmed the invariant
histidines as heme ligands (48). These biochemical
approaches would have been valuable in demonstrating that the four
conserved histidines in A. vinelandii HoxZ are indeed
ligands to the hemes. Difference spectra (reduced minus oxidized) of
A. vinelandii Wt and HoxZ membranes were
examined, but given the large heme background found in this bacterium
(e.g., cytochrome bc1 complex and cytochrome bd oxidase), we were unable to identify the absorption
associated with HoxZ. A. vinelandii hydrogenase, like most
NiFe hydrogenases, was purified as a heterodimer without HoxZ
(42). Our efforts to purify A. vinelandii HoxZ
along with the heterodimer or HoxZ itself were not successful. However,
the current site-specific mutagenesis approach shows that loss of any
of these four ligands results in the complete loss of hydrogenase
activity and supports the conclusion that the four fully conserved
histidines in the hydrogenase b-type cytochrome are ligands
to two hemes.
A tyrosine residue is capable of binding heme and is the natural ligand
in catalase (31) and cytochrome d1 of
nitrite reductase cytochrome cd1 (13)
and serves as a ligand in natural methemoglobin mutants
(35). Three of the four HY A. vinelandii HoxZ
ligand mutants (positions 33, 74, and 208) had the same loss of
hydrogenase activity as the corresponding HA ligand mutants.
However, the mutant with tyrosine in position 194 had ~20% of Wt
activity, suggesting that tyrosine substitutes as a ligand for
histidine in this position but does not enable Wt levels of activity.
The failure to reach Wt activity may indicate that the larger size of
the tyrosine residue prevents optimal conformation and helix bundling,
or perhaps the tyrosine substitution affects redox potential.
Imd has been used with several substituted heme proteins (e.g.,
horseradish peroxidase H170A [31], cytochrome
c peroxidase H175G [24], a soluble version
of heme oxygenase H25A [47], and soluble guanylate
cyclase H105G [50]) to serve as an exogenous ligand
and rescue heme binding. In each of these studies with purified
proteins, the iron in the native protein is five-coordinate with a
single histidine as the proximal ligand and Imd replaced this ligand,
albeit to various degrees. Circular dichroism spectra of E. coli expressing the sperm whale myoglobin gene showed that Imd
rescued myoglobin H93G (4). The present study probed
multiple ligand sites and demonstrated in vivo Imd rescue of one of
these sites in this integral membrane protein. Imd apparently fulfills the structure and function of a ligand in H194A/Y to the same capacity
as histidine. Since hydrogenase function in H194A can be controlled
simply by adding Imd to the growth medium or assay buffer, i.e.,
converting hydrogenase from inactive to active, this provides a system
that could be used for a variety of in vivo hydrogenase studies.
The H194 site in HoxZ is unique in allowing free Imd or a tyrosine
residue to restore activity, but the rationale for this is not clear.
H33 and H74 are the only ligands on helices A and B, respectively, but
194 and 208 are both on helix D. With single replacements, helix D
would be held in place by the unsubstituted ligand. However, the
ability of Imd to function in a site cannot be due solely to being on
helix D, since Imd does not restore O2-dependent
H2 oxidation activity to H208A. H194 is the only site in
HoxZ with a neighboring histidine (H195). In some proteins, such as
A. vinelandii hydrogenase HoxK (41) and
ferredoxin I FdxA (23), a neighboring or free cysteine
residue substituted for a missing cysteine ligand. However, neither
H195 nor its counterpart in W. succinogenes (H187) can
substitute for the ligand (16). Furthermore, the W. succinogenes HydC mutant H187A had Wt activity, which implies that
this neighboring histidine does not play a critical role in Wt
hydrogenase activity. We do not know why Imd and tyrosine can
substitute for this ligand and only this ligand. The other histidine
ligands may have different local environments that do not aid in
positioning or stabilizing a substitution or addition of a free ligand
or are not able to provide the appropriate redox potential for the heme.
In A. vinelandii, when four histidines with some degree of
conservation among 14 hydrogenase cytochrome b homologues
were replaced by alanine or tyrosine, all four mutants were active in
O2-dependent H2 oxidation activity. Three of
these, H97Y, H98A, and H191A, had full Wt O2-dependent
H2 oxidation activity. Substitution of three different
nonligand histidines in HydC of W. succinogenes (16), including a histidine (homologous to A. vinelandii HoxZ H195), produced altered proteins that had Wt
activity. One of the A. vinelandii HoxZ nonligand histidine
mutants, H136A, had only one-third of Wt O2-dependent
H2 oxidation activity. The histidine in this position
apparently plays a role in the structure or function of HoxZ. H136 is
in the BC loop region on the cytoplasmic side of the cytoplasmic
membrane near the hydrophobic core of the membrane and in close
proximity to the high potential heme. Of the surrounding residues, G135
is conserved in most and N137 and P138 are conserved in all HoxZ
homologues examined. Besides heme ligation, other possible roles
suggested for conserved histidines in b-type cytochromes are
functions in quinol binding and modulating heme redox potential (7). H136 in A. vinelandii HoxZ, H217 of the
bc1 complex (Rhodobacter capsulatus
numbering) (15), and H13 of Bacillus subtilis
succinate:ubiquinone oxidoreductase (17) are all located on
the cytoplasmic side of the plasma membrane, and replacement of each of
these nonligand mutants affects activity. Because redox properties of
the quinone in the Qi site were affected, H217 was
suggested to form part of the Qi site binding pocket
(15). H13Y had half the activity of Wt. From electron
paramagnetic spin studies, Hägerhäll et al. (17)
concluded that there was a structural change in the local environment
of the heme, bH.
Influence of alterations in HoxZ on hydrogenase activity of the
HoxKG heterodimer.
To localize the loss of
O2-dependent H2 oxidation activity to the
substitutions in HoxZ, the hydrogenase activity in the mutants was
determined with an assay (H2-dependent MB reduction)
requiring only functional heterodimers. Previous studies (5, 16,
32, 40) showed that the heterodimer is active in mutants in which the cytochrome b component of hydrogenase is disrupted. As
expected, the hydrogenase heterodimers were active in all the HoxZ
mutants (Table 3), which indicated that heterodimer activity could
still occur without a functional HoxZ. However, quantitative assays revealed that the A. vinelandii HoxZ mutants did influence
the heterodimer activity and that the effects depended on the residue substituted and whether or not Imd was included (Fig. 3). H33A/Y had
full Wt activity, while H74A, H194A and H208A had less than Wt
activity. The mechanism by which HoxZ influences the
H2-dependent MB reduction activity of the heterodimer is
uncertain, particularly since MB can bind directly to the heterodimer
(42). Perhaps these three HoxZ ligands, H74, 194, and 208, influence alternative and more efficient pathways of electron transfer
to MB, or perhaps they fulfill a structural role in anchoring the
heterodimer correctly. For H194 and H208, but not H74, Imd restored
this function. Imd appears to influence a structural role in support of
MB reduction by the heterodimer. The HA substitution in position
136, which is not a heme ligand, resulted in ~20% greater
H2-dependent MB reduction and 75% lower
O2-dependent H2 oxidation rate than Wt. If H136
is part of a quinone binding site, perhaps the HA substitution decreases the efficiency of quinone binding while increasing the efficiency of MB binding. Since H136 is on the cytoplasmic side, this
position probably is not involved in heterodimer binding.
Similarities between quinone-interacting proteins (26)
provide insight on how cytochrome b may structurally affect
the other subunits. The presence or absence of hemes in
succinate-ubiquinone oxidoreductase in E. coli determined
whether the heterodimer was able to bind the membrane and function
(30), suggesting that incorporation of the heme resulted in
a conformational change that exposed binding sites for the two subunits
or that the heme may interact directly with them (19).
Several lines of evidence indicate that the cytochrome b
component of hydrogenase can influence the conformation of the
heterodimer. First, in an E. coli deletion mutant of HyaC (a
HoxZ homologue), the heterodimer occurs in three forms, unlike the
single form of Wt. This heterogeneity may result from differential
folding of the heterodimer (28). Second, when an
Alcaligenes eutrophus HoxZ deletion mutant was analyzed for hydrogenase activity via an in-gel nitroblue tetrazolium reduction with
H2 and phenazine methosulfonate, the location of activity in the HoxZ deletion mutant indicated that the heterodimer of HoxZ was in a different conformation than the heterodimer
in Wt (6). Third, ligands binding in positions 74, 194, and
208 in HoxZ of A. vinelandii are apparently structurally
important for full heterodimer activity. Perhaps an Imd-induced
conformational change in HoxZ point mutants in positions 194 and 208 enables the heterodimer to bind to HoxZ in a conformation that allows
the heterodimer to regain full activity.
Results of the present HoxZ study of A. vinelandii indicate
that specific histidine residues 33, 74, 194, and 208 in HoxZ are
required for physiologically functional electron transfer. Based on
this requirement, the intramolecular locations of the residues, and
their conservation among HoxZ homologues, we conclude that these
residues are ligands to two hemes. Residues 74, 194, and 208 are also
needed structurally for full catalytic activity of the heterodimer even
in the absence of electron flow through HoxZ. The four heme ligand
sites in A. vinelandii HoxZ are unique with regard to their
chemical tolerance to residue substitution and small molecule
complementation, as measured by two hydrogenase assays
(O2-dependent H2 oxidation and
H2-dependent MB reduction). One nonligand histidine, H136,
is also important to hydrogenase in physiological electron transport.
Both ligands near the periplasmic side of the plasma membrane, those
nearest the heterodimer, and one of the ligands on the cytoplasmic side
are required for optimal heterodimer catalytic activity. Thus, in the
hydrogenase of A. vinelandii, the ligands in the
b-type cytochrome HoxZ are required structurally and
functionally for optimal H2 oxidation activity. Several
roles have been explored for HoxZ and its homologues (6, 8, 16,
20, 26, 32, 40), including mediating electron flow from
hydrogenase to the respiratory chain. The present results identify
specific histidine residues in HoxZ involved in this role.
This work was supported by U.S. Department of Energy grant
FG06-90ER20013 to D.J.A. and made possible in part by a grant from the
AAUW (American Association of University Women) Educational Foundation.
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Abstract
Introduction
Present address: Division of Hematology, Washington University
School of Medicine, St. Louis, MO 63110.
