From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 and the Universitätsklinikum Frankfurt, ZBC, Institut für Biochemie I, D-60590 Frankfurt, Germany
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ABSTRACT |
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The crystal structure of the bovine Rieske
iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur
cluster and the sulfur atom (S) of a cysteine
residue that coordinates one of the iron atoms form hydrogen bonds with
the hydroxyl groups of Ser-163 and Tyr-165, respectively. We have
altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces
cerevisiae Rieske iron-sulfur protein by site-directed
mutagenesis of the iron-sulfur protein gene to examine how these
hydrogen bonds affect the midpoint potential of the iron-sulfur cluster
and how changes in the midpoint potential affect the activity of the
enzyme.
Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1
of the cluster lowers the midpoint potential of the cluster by 130 mV,
and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to
S of Cys-159 lowers the midpoint potential by 65 mV.
Eliminating both hydrogen bonds has an approximately additive effect,
lowering the midpoint potential by 180 mV. Thus, these hydrogen bonds
contribute significantly to the positive midpoint potential of the
cluster but are not essential for its assembly. The activity of the
bc1 complex decreases with the decrease in
midpoint potential, confirming that oxidation of ubiquinol by the
iron-sulfur protein is the rate-limiting partial reaction in the
bc1 complex, and that the rate of this reaction
is extensively influenced by the midpoint potential of the iron-sulfur
cluster.
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INTRODUCTION |
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The Rieske iron-sulfur protein is a ubiquitous component of
cytochrome bc1 complexes (1-4) and has been
shown to be essential for electron transfer and energy transduction by
purification of the protein in a reconstitutively active form and
reconstitution to iron-sulfur protein depleted
bc1 complex (5, 6). The electronic environment
of the [2Fe-2S] cluster in the Rieske iron-sulfur protein differs
from that in plant type [2Fe-2S] ferredoxins as evidenced by a
distinct EPR spectrum (1) and a redox midpoint potential of the Rieske
protein (e.g. +280 mV) that is much more positive than the
midpoint potentials typical of the ferredoxins (e.g. 420
mV; Ref. 2). The high midpoint potential of the iron-sulfur cluster is
essential for the function of the Rieske protein in the Q cycle
mechanism of the bc1 complex (7, 8), in which
the Rieske protein is the primary electron acceptor and drives the
electron transfer reaction by oxidizing ubiquinol and divergently
transferring one electron to cytochrome c1,
while the ubisemiquinone that is formed from ubiquinol reduces the low potential b heme.
Recently, the crystal structure of the water-soluble part of the Rieske
iron-sulfur protein of bovine heart mitochondrial bc1 complex has been elucidated at 1.5 Å (9,
10). Ten strands form three layers of anti-parallel
sheets in a
flat spherical molecule as shown in Fig. 1A. The cluster
binding fold is a small domain-like structure comprising approximately
46 residues; it consists of a distorted four-stranded antiparallel
-sheet and three loops. The loops between the strands
4-
5 and
6-
7 each contribute one cysteine and one histidine ligand to the
cluster. Cysteine 139 and 158 are ligands of Fe-1 and are buried within the protein. Histidines 141 and 161 are ligands to the redox-active Fe-2 and are completely exposed to the solvent. The loops are cross-linked by a disulfide bridge formed by two additional cysteine residues in each loop. This disulfide bridge forms one side of the
cluster binding fold and stabilizes it. The other side of the cluster
binding fold is covered by the so called "Pro loop."
From the crystal structure several factors were identified, which
contribute to the positive midpoint potential of the Rieske protein,
including a net charge of 0/1 for the oxidized and reduced iron-sulfur cluster, the electronegativity of the histidine ligands, and the proximity of Fe-2 to the surface of the protein. The midpoint potential should also be affected by the complex hydrogen bond network
through which the iron-sulfur cluster is linked to the surrounding
protein. With the exception of Cys-158, which has only one hydrogen
bond to the nitrogen of Cys-160, all sulfur atoms are involved in two
hydrogen bonds each.
The hydrogen bond network to sulfur atoms includes Ser-163 and Tyr-165.
Both residues are located in strand 7 and are conserved in all
Rieske iron-sulfur proteins that oxidize ubiquinol and that have been
sequenced to date. As shown in Fig.
1B, the hydroxyl group of
Ser-163 is hydrogen-bonded to S-1, at 3.2 Å. The hydroxyl group of
Tyr-165 is hydrogen-bonded to the S
of Cys-139, a ligand
of Fe-1, at 3.1 Å. Both residues are likely to contribute to the
positive midpoint potential of the Rieske protein by decreasing the
negative charge density around the sulfur atoms through their respective hydrogen bonds.
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In the present study we have examined the effects of the hydrogen bonds from the hydroxyl of Ser-163 to the internal sulfur atom and from the hydroxyl of Tyr-165 to the sulfur of one of the cysteine ligands on the midpoint potential of the Rieske iron-sulfur cluster by introducing amino acid changes at equivalent positions (Ser-183 and Tyr-185) in the yeast iron-sulfur protein through site-directed mutagenesis of the cloned gene. We show that conservative substitutions that eliminate these two hydrogen bonds lower the midpoint potential, without affecting the stability of the protein, and that these changes are accompanied by decreases in turnover numbers of the bc1 complexes containing the altered forms of the Rieske protein.
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EXPERIMENTAL PROCEDURES |
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Materials-- SDS, acrylamide, and bisacrylamide were from Bio-Rad. Urea and agarose (Ultra Pure) were from Life Technologies, Inc. DNA was isolated from agarose gels using the QIAquick gel extraction kit from QIAGEN. NytranTM nylon membranes were from Schleicher & Schuell. Glass beads (0.5 mm) were obtained from Biospec Products. Horse heart cytochrome c and the ubiquinone analog 2,3-dimethoxy-5-methyl-6-n-decyl-1-4-benzoquinone were from Sigma. Yeast extract, peptone, bacto-agar, and tryptone were from Difco.
Site-directed Mutagenesis-- The plasmid pGEM3-RIP1, carrying the wild type Saccharomyces cerevisiae Rieske iron-sulfur protein gene, was used for site-directed mutagenesis with the CLONTECH Transformer mutagenesis kit (11). The mutational changes were confirmed by automated sequencing using the Dye Deoxy terminator cycle sequencing kit from Applied Biosystems Inc. A PstI/SacI fragment from the mutated pGEM3-RIP1 was then subcloned into the single-copy yeast vector pFL39-RIP1, from which this fragment was removed. Cells of the RIP1 deletion strain JPJ1 were then transformed with the pFL39-RIP1 plasmid carrying the site-directed mutations (12).
Western Analysis of Mitochondrial Membranes-- Yeast cells were grown 2-3 days on synthetic dextrose medium lacking tryptophan. Mitochondrial membranes were isolated as described previously (13). Protein concentrations were determined by a modified Lowry method (14, 15). Membranes were resolved by SDS-polyacrylamide gel electrophoresis (16), and iron-sulfur protein and cytochrome c1 were detected by Western blotting (17) using monoclonal antibodies to yeast iron-sulfur protein and cytochrome c1 (13).
Purification of Cytochrome bc1 Complexes-- For all of the yeast mutants in which the iron-sulfur protein was detectable by Western blotting of the mitochondrial membranes, the cytochrome bc1 complexes were purified in two different laboratories and by two different methods. In one laboratory the membranes were extracted with dodecyl maltoside and the complexes purified by ion-exchange chromatography (18), and in the other laboratory the membranes were extracted with Triton X-100 and purified by hydroxyapatite chromatography (19).
Optical Spectroscopy and Enzyme Assay--
Ubiquinol-cytochrome
c oxidoreductase activities of mitochondrial membranes and
purified cytochrome bc1 complexes were assayed at pH 7.0 and 23 °C using 50 µM
2,3-dimethoxy-5-methyl-6-n-decyl-1-4-benzoquinol as
substrate and 50 µM horse heart cytochrome c
(5). Reduction of cytochrome c was monitored in an Aminco
DW-2A spectrophotometer at 550 versus 539 nm in dual
wavelength mode. Turnover numbers of the bc1
complexes in situ and of the purified enzymes were calculated on the basis of the concentration of cytochrome
b, which was determined from optical spectra of the
dithionite reduced minus ferricyanide oxidized samples (20). Assays
were performed in triplicate, and turnover numbers are expressed as
percent of the turnover number of the bc1
complex from the wild-type strain. Turnover numbers of the enzyme from
the wild-type strain were 200-220 s1 as reported
previously (18).
CD-monitored Redox Titration of Isolated bc1
Complex--
CD-monitored redox titrations of isolated cytochrome
bc1 complexes were performed in an optically
transparent thin layer cell with a path length of 0.1 mm built in-house
and using a Jasco J-720 spectropolarimeter as described previously
(21). The transparent electrode was a pyridine-3-carboxaldehyde
thiosemicarbazone modified gold grid with a mesh size of 500 wires/inch
and a transmission of 60% (Buckbee-Mears, St. Paul, MN). The potential
was controlled through a Wenking MP 95 potentiostat (Bank Elektronik,
Clausthal, Germany). To facilitate redox equilibration, methylviologen
(E° = 449 mV), anthraquinone-2-sulfonate
(E° =
225 mV), 2-hydroxy-1,4-napthoquinone (E° =
145 mV), menadione (E° =
13 mV),
duroquinone (E° = +5 mV), phenazine ethosulfate
(E° = +55 mV), phenazine methosulfate (E° = +80 mV), trimethyl-1,4-benzoquinone (E° = +99 mV),
1,2-napthoquinone (E° = +144 mV), 2,6-dichlorophenol
indophenol (E° = +217 mV), N,N,N',N'-tetramethyl-p-phenylenediamine
(E° = +276 mV), and ferricyanide (E° = +408
mV) were added at final concentrations of 25 µM each to
approximately 0.5 mM cytochrome bc1
complex in 100 mM NaCl, 35 mM
MOPS,1 0.025% Triton X-100,
pH 7.0. Neither the oxidized nor the reduced form of the mediators
showed any CD signal, even at 10-fold higher concentrations. All
potentials have been recalculated with reference to the standard
hydrogen electrode.
Crystal Structure Calculations and Graphics--
Views of the
iron-sulfur protein were generated from the crystal structure
coordinates of the bovine protein using the molecular graphics program
RasMac, version 2.6 (Roger Sayle). Inter-atomic distances were
measured, and rotamer positions of the various amino acid substitutions
were examined with Swiss PDB-viewer, version 2.2 (Glaxo Institute of
Molecular Biology, Geneva, Switzerland). Although this program does not
calculate true energy minimization's for substituted amino acids, it
does calculate a "score" for each rotamer position of a substituted
amino acid according to the empirical formula S = 4X + 3Y + 2Z nH,
where X is the number of clashes with backbone nitrogen or
carbon atoms, including the
carbon, Y is the number of
clashes with backbone oxygen atoms, Z is the number of
clashes with side-chain atoms, and H is the number of
potential hydrogen bonds.
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RESULTS |
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Choice of Mutations-- The amino acid sequences of the bovine and yeast Rieske iron-sulfur proteins are almost identical throughout the region containing the cluster ligands and the "Pro loop" as shown in Fig. 2. The numbering of the two sequences differ by 20, so that Ser-163 in the bovine iron-sulfur protein equals Ser-183 in yeast, and Tyr-165 in the bovine protein equals Tyr-185 in yeast.2 Only 6 of the 44 amino acids in the region surrounding the iron-sulfur cluster are not identical in the bovine and yeast proteins, and 5 of these are conservative changes. The only non-conservative change is Glu-169 in the yeast protein versus Asn-149 in the bovine protein. In the bovine crystal structure, this residue is on the surface of the protein, in the loop that extends from the right of the protein in the orientation shown in Fig. 1, and distal from the cluster. It is thus unlikely that this single non-conservative change results in any substantive difference between the boine and yeast proteins.
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Growth Characteristics and Ubiquinol-Cytochrome c Reductase
Activities--
To monitor effects of the RIP1 mutations on
respiratory activity, transformants were grown on solid medium
containing ethanol/glycerol at 25, 30, and 37 °C (Table
I). Only three mutants, carrying the
S183T, Y185F, and Y185W mutations, had sufficient respiratory activity
to allow growth (Pet+) at all three temperatures. The other
mutations resulted in a petite phenotype
(pet), such that the yeast were unable to grow
on ethanol/glycerol at any of the three temperatures. When grown on
ethanol/glycerol at 30 °C in liquid culture, the doubling time of
the wild-type strain was 3.6 h, and the doubling times of the
mutants with the S183T, Y185F, and Y185W forms of the iron-sulfur
protein increased to 4.0, 4.4, and 9.2 h, respectively.
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Western Blot Analysis of Mitochondrial Membranes from Yeast Strains with Altered Rieske Proteins-- Mitochondrial membranes of JPJ1 expressing the mutated genes from pFL39-RIP1 were assayed for their content of iron-sulfur protein by Western blotting with monoclonal antibodies to iron-sulfur protein, using cytochrome c1 as an internal control to establish that equal amounts of mitochondrial protein were loaded onto the gel. As expected, the deletion strain JPJ1 transformed with the pFL39 plasmid lacking any RIP1 gene showed no immunodetectable Rieske protein (Fig. 4).
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Determination of Midpoint Potential of Mutated Iron-Sulfur Protein Using Circular Dichroism-- In the reduced state the Rieske protein displays a strong negative CD band at 500 nm, which is free of interfering signals in isolated bc1 complex (21). We isolated cytochrome bc1 complexes from yeast expressing selected mutated forms of the iron-sulfur protein and monitored the reduction status of the iron-sulfur cluster by CD spectroscopy as the applied redox potential was varied (Fig. 5). From these titrations, we determined the midpoint potentials of the [2Fe-2S] clusters, as shown in Fig. 6.
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Effects of Mutational Changes on the Structure of the Iron-Sulfur Protein-- In addition to allowing determination of the midpoint potential, CD absorption arising from the iron-sulfur cluster monitors coupling of the iron-sulfur cluster to the chiral protein and the non-planar geometry that depends on the protein environment. The CD spectra in Fig. 5 show that, with the exception of the bc1 complex containing the S183C form of the iron-sulfur protein, in which the iron-sulfur cluster is missing, the shape and intensity of the signature resonance from the iron-sulfur cluster in the 500 nm region is not significantly altered by the Y185F, Y185W, S183T, S183A, or Y185F,S183A mutations. The spectra are good evidence that the conservative changes in the altered forms of the iron-sulfur protein in which the cluster has been retained have not caused any structural rearrangement in the protein environment surrounding the iron-sulfur cluster.
This is further supported by the EPR spectra of the purified bc1 complexes containing these altered forms of the Rieske protein, which showed small variations of all three principal g values (gz, gy: <0.01; gx: <0.04) but retained the characteristic features of "Rieske" clusters as well as their intensity (results not shown). These spectra indicate small perturbations of the electronic distribution within the cluster, as would be expected if the hydrogen bonds from Ser-183 and Tyr-185 participate in electron delocalization, but exclude significant structural perturbations. Optical spectra of the cytochromes provide a sensitive monitor of the interaction between the domain of the iron-sulfur protein that includes the iron-sulfur cluster and the domain of cytochrome b that includes the low potential b heme at center P. In mutants where the iron-sulfur cluster is absent due to mutational changes that eliminate one of the liganding histidines or cysteines (13) or where the iron-sulfur cluster is not stably inserted due to deletion of a supernumerary subunit of the bc1 complex (cf. Fig. 1 in Ref. 25), there is a loss of absorption in the 560-562-nm region of the optical spectrum, presumably due to indirect effects on the cytochrome b environment resulting from these changes in the iron-sulfur protein. The optical spectra in Fig. 5 show that no such changes are evident in the purified bc1 complexes containing mutated iron-sulfur proteins. Perhaps the best evidence that the conservative mutational changes have not altered the structure of the protein is that the protein remains stably associated with the cytochrome bc1 complex and the iron-sulfur cluster remains intact during purification, which involves detergent extraction and ion-exchange (18) or hydroxyapatite (19) chromatography, as indicated by the fact that there was no decrease in turnover of the complex as a result of the purifications. The apparent lack of structural changes in the protein environment surrounding the iron-sulfur cluster is consistent with the highly constrained nature of the iron-sulfur fold as revealed by the crystal structure (10).Relationship between Midpoint Potential of the Rieske Iron-Sulfur Protein and Activity of the Cytochrome bc1 Complex-- Ubiquinol-cytochrome c reductase assays were run under conditions such that the reaction is zero order with respect to quinol and cytochrome c; consequently, the rate of cytochrome c reduction reflects the rate of electron transfer within the enzyme. As shown in Fig. 7, there is an exponential relationship between ubiquinol-cytochrome c reductase activity and the midpoint potential of the Rieske iron-sulfur protein as the latter varies from +100 to +280 mV. In the range between 220 and 280 mV, the rate increases 2.5-fold with an increase of 60 mV. As discussed below, this confirms that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc1 complex (26) and demonstrates the extent to which the rate of electron transfer from bound quinol to cytochrome c1 is controlled by the midpoint potential of the iron-sulfur cluster.
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DISCUSSION |
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The Rieske iron-sulfur protein has a redox potential that is about 400-700 mV higher than the redox potentials of other [2Fe-2S] proteins. The crystal structure of the protein shows that the iron-sulfur cluster is embedded in a complex hydrogen bond network. We expected that the hydrogen bond network should contribute to the positive midpoint potential. To study the effects of the hydrogen bond network on the redox potential of the iron-sulfur protein, we introduced replacements for Ser-183 and Tyr-185, which contribute two of the seven hydrogen bonds of this network, by site-directed mutagenesis of the cloned iron-sulfur protein gene.
The most striking result from this mutational analysis is the finding
that two hydrogen bonds formed by these two residues increase the
midpoint potential of the Rieske [2Fe-2S] cluster by approximately
180 mV, with the hydrogen bond from the Ser-183 hydroxyl group to S-1
of the iron-sulfur cluster contributing 130 mV and the hydrogen bond
from the Tyr-185 hydroxyl group to S of Cys-159 contributing 65 mV.
The presence of these two hydrogen bonds in the wild-type protein would
decrease the electron density in the cluster and facilitate
delocalization of the electron in the cluster during reduction of Fe-2.
The potential of the iron-sulfur protein with the S183A mutation is
comparable to that observed in menaquinol oxidizing bc
complexes (27), where alanine instead of serine has been found in the
native sequences (28).
Substitution of Ser-183 with Thr resulted in retention of 80% of the wild-type bc1 activity, while substitution of Ser-183 with Ala resulted in only 10% of wild-type bc1 activity (Table I). Since modeling these substitutions indicated that the rotamer scores for Ser, Thr, and Ala were identical, we deduce that the S183T substitution retained the hydrogen bond to S-1. The slight decrease of the redox potential in the S183T form of the protein is probably due to some subtle steric distortion resulting from the additional methyl group in the side chain of the Thr residue. This would alter the length and or angle of the hydrogen bond from the Thr hydroxyl to S-1, resulting in a weakening of the hydrogen bond, although this was not obvious when the Thr rotamers were viewed in the computer program. A previously described yeast mutant (22) in which Ser-183 of the Rieske protein is replaced by Leu showed significantly less immunologically detectable iron-sulfur protein, suggesting that insertion of a bulkier side chain results in an unstable protein.
Replacement of Ser-183 with Cys resulted in a Rieske protein lacking iron-sulfur cluster, although the apoprotein was present in essentially normal amounts (Fig. 4). This is the first example of a mutation in the Rieske iron-sulfur protein where the protein appears fully stable in the absence of iron-sulfur cluster. Since the Ser-183 hydroxyl group is not essential for formation of the cluster, as evidenced by the presence of the cluster in the S183A form of the protein, and the rotamer scores for the Cys substitution were not significantly different than for Ser, we conclude that the presence of an additional Cys residue interferes with cluster formation, possibly at the point of iron insertion.
Among the eight substitutions for Tyr-185, only Y185F and Y185W
resulted in functionally active iron-sulfur protein. The change in
midpoint potential was slightly greater in the Y185W form of the
protein (+195 mV) than in the Y185F form (+217 mV). We considered the
possibility that the Y185W mutation might have allowed an alternative
hydrogen bond from the indol nitrogen to the S of Cys-159, resulting
in an additional decrease in the midpoint potential (Fig. 4). However,
modeling of the Trp substitution indicates that, of the 15 possible
rotamers, those with the lowest two energies only bring the indol
nitrogen within 5.02 and 5.51 Å of the S
of Cys-159, and none of
the rotamers bring the indol nitrogen less than 4.40 Å from the
sulfur. The difference between the Trp and Phe substitutions is thus
probably due to distortions of the cluster environment resulting from
the introduction of the bulkier indol ring in comparison to the phenyl
group. Consistent with this interpretation, we observed that the
iron-sulfur cluster in the Y185W form of the protein was not stable
during freezing of the mitochondrial membranes or the isolated
cytochrome bc1 complex, whereas the protein with
the Y185F substitution was
stable.3
Yeast mutants in which Tyr-185 of the Rieske protein was replaced with His, Leu, Thr, Ser, Arg, or Asp were only able to grow fermentatively. Western blotting of mitochondrial membranes established that the amount of Rieske protein was drastically diminished in the yeast carrying the Y185R mutation and undetectable in membranes from the yeast with the Y185H, Y185L, Y185T, Y185S, and Y185D forms of the protein (Fig. 4). The absence of Rieske iron-sulfur protein in the yeast with the Y185H, Y185L, Y185T, Y185S, and Y185D iron-sulfur protein mutations is not simply due to failure to insert iron-sulfur cluster or a secondary consequence of the loss of an unstable iron-sulfur cluster, since the mature apoprotein was present in the yeast with the S183C form of the protein (Fig. 4), for which the CD spectrum indicated the cluster was absent. Likewise, in a previous study, immunologically detectable iron-sulfur protein was also present, although in decreased amounts, in the mitochondrial membranes of yeast mutants in which the Cys and His ligands required for formation of the [2Fe-2S] cluster were eliminated by site-directed mutagenesis (13). The conservative changes in the protein introduced by these mutations thus differ markedly from the mutation characterized by Gatti and co-workers (22), where only a small change in midpoint potential (50 mV) was accompanied by instability of the protein and significant loss of iron-sulfur cluster.
The lack of immunologically detectable Rieske protein in mitochondrial membranes from yeast with the Y185H and Y185D forms of the protein is somewhat surprising, since the profile of rotamer scores indicates that there are more stable rotamers for these substitutions than for the wild-type protein. One possible explanation for the absence of protein in these mutants is that the imidazole nitrogens of the histidine and carboxyl groups of the aspartate form hydrogen bonds during the protein folding process that result in aberrantly folded protein, which is then degraded. An additional source of instability in the Y185D form of the protein is that the carboxylate anion may be excluded from the otherwise hydrophobic environment surrounding Tyr-185 during protein folding.
The absence of stably formed Rieske protein in the yeast with the Y185L, Y185T, and Y185S forms of the protein can be explained by the large number of prohibited rotamers, in the case of Y185L, and by an unstable cavity proximal to the Cys-159 and Cys-178 residues that coordinate the cluster (see Fig. 1B) when the small Thr and Ser residues are substituted for the bulkier Tyr. From modeling these substitutions, the resulting protein instability appears to be because it is necessary to maintain the tight packing of the cluster binding fold, which is thought to protect the protein against attack by proteases (10).
The use of Western blots to screen for the presence of the Rieske protein allowed mutations resulting in an unstable protein to be recognized. The Western blots also reveal an important aspect of iron-sulfur protein assembly. In S. cerevisiae mitochondria, the Rieske iron-sulfur protein is post-translationally processed in two steps by two peptidases during import and assembly into the cytochrome bc1 complex (29). The intermediate iron-sulfur protein (i-ISP) that is formed during the first step of this processing is normally detectable in only small amounts in mitochondrial membranes or purified cytochrome bc1 complex (Fig. 4; see also Fig. 5 in Ref. 23). In the current study, we have observed that significant amounts of incompletely processed i-ISP are visible in mitochondrial membranes from the mutants with both the S183A and S183C forms of the protein (Fig. 4). This species, which migrates slightly more slowly than the mature sized protein, was previously identified as i-ISP by N-terminal sequencing of the protein (23). Since i-ISP does not accumulate in mitochondrial membranes of any of the yeast mutants with changes at Tyr-185 of the Rieske protein and in which the protein is immunologically detectable, we conclude that the accumulation of i-ISP in the mitochondrial membranes of the yeast with the mutations at Ser-183 reflects a facilitative effect of the Ser-163 hydroxyl group on formation of the [2Fe-2S] cluster. The accumulation of i-ISP in the yeast with the replacements at Ser-183 is evidence that insertion of the cluster precedes conversion of i-ISP to mature iron-sulfur protein, but is not obligatory for this final step in Rieske iron-sulfur protein maturation (13).
The relationship between ubiquinol-cytochrome c reductase activity and Rieske midpoint potential confirms that oxidation of ubiquinol by the Rieske protein is the rate-limiting partial reaction in the bc1 complex when the enzyme is operating catalytically (26). To illustrate this, we have depicted electron transfer from ubiquinol to cytochrome c through the Q cycle mechanism as consisting of seven electron transfer steps in the bc1 complex in Fig. 8. Since the cytochrome c reductase assays are performed under conditions where the reaction is zero order with respect to cytochrome c, reaction 6 can not be limiting. If any of the iron-sulfur protein independent electron transfer steps within the Q cycle, reactions 1b, 3, 4, or 5, were significantly slower than those involving the iron-sulfur protein, reactions 1a and 2, changing the potential of the iron-sulfur cluster would not alter the activity of the bc1 complex. Thus, electron transfer from ubiquinol to iron-sulfur protein or from iron-sulfur protein to cytochrome c1 must be the rate-limiting partial reaction within the enzyme.
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The changes in midpoint potential of the iron-sulfur protein could affect either or both of two electron transfer reactions, from bound quinol to the iron-sulfur cluster (see Fig. 8; reaction 1a) or from the iron-sulfur cluster to cytochrome c1 (reaction 2). Since the rate decreases as the potential decreases, and there is no theoretical basis on which to explain how increasing the increment in potential between the iron-sulfur cluster and cytochrome c1 could lead to a decrease in electron transfer rate, the decline in rate that accompanies the decrease in iron-sulfur cluster potential must reflect a decrease in the rate of electron transfer from ubiquinol to the iron-sulfur cluster.
Between 220 and 280 mV, the turnover number of the enzyme increases 2.5-fold as the midpoint potential of the iron-sulfur cluster increases 60 mV. If the rate dependence on midpoint potential is predictable from Marcus electron transfer theory, it would be expected to change 3.1-fold for a 60-mV change in potential. The less than predicted change indicates that under conditions where the activity is zero order with respect to ubiquinol and cytochrome c, the activity of the bc1 complex is influenced extensively, although not solely, by the midpoint potential of the iron-sulfur cluster. If deprotonation of ubiquinol is prerequisite to electron transfer (30), the protonic chemistry must be sufficiently fast as not to impede significantly the potential dependence of the cytochrome c reductase rate.
Since the decline in ubiquinol-cytochrome c reductase rate
with declining midpoint potential of the Rieske protein results from a
decline in the rate of electron transfer from ubiquinol to the
iron-sulfur cluster, it is not surprising that the rate approaches zero
as the midpoint potential of the Rieske protein approaches 100 mv,
since the midpoint potential of the ubiquinol/ubiquinone couple is
approximately 100 mV (31). The fact that the rate does not continue to
decline in a linear manner below 220 mV and reach zero at approximately
160 mV suggests that a second parameter becomes operative at limiting
values of Rieske protein potential. One such possibility is that rapid
electron transfer from the Rieske iron-sulfur protein to cytochrome
c1, for which the rate has been estimated to be
greater than 5 × 105 s1 (32), maintains
the Rieske iron-sulfur clusters in these mutants in a highly oxidized
state, so that the operative redox potentials are higher than the
midpoint potentials determined from equilibrium titrations. A second
possibility is that this results from the concerted nature of the
divergent two-electron transfer reaction at center P (reactions 1a and
1b in Fig. 8), that involves contributions from a domain on cytochrome
b, in addition to the Rieske protein (8).
The changes of the redox potential of the iron-cluster should affect the population of the semiquinone intermediate at center P, which, according to the "proton-gated affinity change" mechanism, is stabilized by the reduced iron-sulfur cluster (33). The non-linear dependence of electron transfer rate on redox potential is consistent with this interpretation, but a more detailed characterization of the kinetics of the bc1 complexes in these mutants will be required to unravel this aspect of the mechanism of ubiquinol oxidation.
Although 30% of the normal ubiquinol-cytochrome c reductase activity in the yeast mutant with a Y185W replacement in the Rieske iron-sulfur protein was sufficient for the yeast to grow on non-fermentable carbon sources, 10% of normal bc1 activity in the mutant with a S183A replacement in the iron-sulfur protein was not sufficient to allow growth on non-fermentable carbon sources. The activities of the bc1 complexes in these two mutants thus bracket a threshold activity between 10 and 30% of that present in wild-type yeast required to support respiratory growth. A threshold of approximately 15% of wild type activity was observed in studies of revertants of yeast mutants deficient in cytochrome oxidase activity.4 Since the boundary activities of 10-30% are linked to midpoint potentials of +155 and +195 mV, respectively, they indicate a critical midpoint potential of the Rieske iron-sulfur protein required to support respiratory growth. Notably, there are no examples of a respiratory competent yeast in which the midpoint potential of the Rieske iron-sulfur protein is below 160 mV (22).
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Note Added in Proof |
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Results simular to those reported here have been obtained in Paracoccus denitrificans, T. Schroeter, O. M. Hatzfeld, S. Gemeinhardt, M. Korn, T. Friedrich, B. Ludwig and T. A. Link, submitted for publication.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 20379 and by Deutsche Forschungsgemeinschaft (DFG) Priority Programme "Transition Metals in Biology and their Coordination Chemistry" (Li 474).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Dartmouth Medical School, 7200 Vail, Hanover, NH 03755. Tel.:
603-650-1621; Fax: 603-650-1389.
1 1 The abbreviations used are: MOPS, 3-(N-morpholino)propanesulfonic acid; i-ISP, intermediate iron-sulfur protein.
2 Hereafter, unless explicitly stated otherwise, we will use the numbering system for yeast.
3 T. Merbitz-Zahradnik, O. M. Hatzfeld, and T. A. Link, unpublished results.
4 C. Ortwein, B. Meunier, A. M. Colson-Corbisier, and T. A. Link, unpublished results.
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