The petite phenotype resulting from a truncated copy of subunit 6 results from loss of assembly of the cytochrome bc1 complex and can be suppressed by overexpression of subunit 9.

Disruption of the gene for subunit 6 of the yeast cytochrome bc1 complex (QCR6) causes a temperature-sensitive petite phenotype in contrast to deletion of the coding region of QCR6, which shows no growth defect. Mitochondria from the petite strain carrying the disruption allele were devoid of ubiquinol-cytochrome c oxidoreductase activity but retained cytochrome c oxidase and oligomycin-sensitive ATPase activities. Optical spectra of cytochromes in mitochondrial membranes from the petite strain lacked a cytochrome b absorption band and had a reduced amount of cytochrome c1. Analysis of mitochondrial translation products showed normal synthesis of cytochrome b. Western analysis of mitochondrial membranes from this disruption strain indicates core protein 1 of the cytochrome bc1 complex is present in normal amounts, while cytochrome c1, the Rieske iron-sulfur protein, subunit 6, and subunit 7 were absent or present in very low amounts. Taken together, these findings indicate a loss of assembly of the cytochrome bc1 complex. High copy suppressors of the disruption strain were selected. Two separate families of suppressors were found. The first contained QCR6. The second family consisted of overlapping clones of a second gene distinct from QCR6. These plasmids contained QCR9, the gene which codes for subunit 9 of the yeast cytochrome bc1 complex. Suppression of the QCR6 disruption strain by overexpression of QCR9 indicates a critical interaction between these two proteins in the assembly of the cytochrome bc1 complex.

Rhodospirillium rubrum (5). The functions of the supernumerary polypeptides in eukaryotes are generally unknown.
Subunit 6 of the yeast cytochrome bcl complex, an unusually acidic protein that has been conserved from yeast to humans (6, 7), is involved in the binding of cytochrome c in cooperation with cytochrome c1 (8). Recently we showed that subunit 6 regulates half of the sites' activity of the dimeric bcl complex, possibly in response to membrane potential (9).
QCR6,' the nuclear gene encoding subunit 6, was cloned and characterized by van Loon and co-workers (6). Several groups have shown that null mutations of subunit 6 have no detectable effect on growth of the yeast on nonfermentable carbon sources (9,lO-12). We report here the characterization of a unique mutant of subunit 6 created by a fortuitous disruption of the gene. This mutation causes the yeast to be a temperature-sensitive petite. We describe the biochemical characterization of the defect in this strain and show that the petite phenotype results from failure to assemble the cytochrome bcl complex. In addition we have selected for high copy plasmid suppressors of the qcr6 petite phenotype and discovered an apparent interaction between subunits 6 and 9 of the bcl complex.

EXPERIMENTAL PROCEDURES
Materials-L-Amino acids, uracil, adenine sulfate, galactose, ampicillin, raffinose, lysozyme, indoleacrylic acid, and ATP were obtained from Sigma. Phenol was purchased from Mallinckrodt Chemical Works. Yeast extract, peptone, Tryptone, and yeast nitrogen base without amino acids were from Difco. Restriction enzymes and T4-DNA ligase were purchased from New England Biolabs or Betbesda Research Laboratories. Nylon membranes were purchased from ICN K & K Laboratories Inc. Nitrocellulose was from Schleicher &
RNA Preparation and Northern Analysis-Yeast RNA was prepared by a method recently developed in this laboratory (15). Northern analysis of QCR6 transcripts was performed essentially as described by Maniatis et al. (16) with the following modifications. RNA from the separating gel was transferred to a nylon membrane. The membrane was prehybridized and hybridized in 50% formamide, 0.1% SDS, 5 X SSPE (0.15 M NaCl, 0.01 M NaH,PO,, 0.001 m EDTA, pH 7.4), 250 pg/ml denatured salmon sperm DNA at 42 "C. The membrane was then washed 3 times for 5 min in 1 X SSPE, 0.1% SDS at 24 "C, followed by washing 6 times for 20 min in 0.1 X SSPE, 0.1% SDS at 65 "C.
Construction of qcrGal:URA3 and qcr6a2:URA3 Strains-The plasmid M13mp8 (730-bp Sau3A:qcrG) contains a 730-bp Sau3Al fragment of the 5' half of QCR6 in an M13 sequencing vector. pUC18:URA3#211 was constructed by placing a 1180-bp HindIII fragment of URA3 into the HindIII site of pUC18, a gift of J. Hill, Albert Einstein Medical College. The insert was removed from M13mp8 (730-bp Sau3Aqcr6) with EcoRI and ligated into the EcoRI site of the plasmid pUC18:URA3#211. The resultant plasmid is named pMES7. The plasmid pMES7 was digested with XbaI and SpeI leaving equivalent sticky ends. The region between these two sites was removed, and the plasmid was religated to create pMES8. pMES8 contains a 210-bp internal fragment of QCRG stretching from the SpeI site to the Sau3Al site.
The plasmid pMES8 was linearized at a unique SacII site in its 210-bp insert. Upon transformation of a u r d -strain of yeast, the plasmid integrates at the SacII site in the yeast genome by homologous recombination (17). After integration the gene for QCR6 is split with one half potentially coding for a protein truncated at the C terminus and the other half potentially coding for a protein truncated at the N terminus. This allele of QCR6 is named qcrGal:URA3. The qcr6a2:uRA3 strain was created in the identical way except that yeast transformants were subsequently screened for stability of the URA3 marker. One such stable transformant was found that contained two tandem repeats of the pMES8 plasmid in the SacII site of QCR6. The genomic arrangement of both of these strains was confirmed by extensive Southern mapping. Both of the strains were petite when grown at 37 "C.
Yeast Plasmid Recouery-Yeast plasmids were purified essentially as described by Hoffman and Winston (18). Following purification, the plasmids were transformed into E. coli strain DH5-a.
Construction of a TrpE:QCRG Fusion Vector, pMES29-The plasmid pATH2 was obtained from Alex Tzagoloff, Columbia University, NY. pATH2 contains the E. coli TrpE gene with a multicloning site at its C terminus in front of a highly inducible promoter (19). pATH2 was opened with XbaI and SalI, and a 1.4-kb SpeIISalI fragment containing most of the coding region for QCR6 was inserted. The resultant plasmid, pMES29, contained an open reading frame in which 145 amino acids of QCRG were fused to the C terminus of TrpE.
TrpE Fusion Protein Production and Affinity Purification of Subunit 6 Antibodies-TrpE protein and TrpE fusion protein were produced essentially as described by Myers et al. (19). A colony of E. coli strain RR1 (F-, hsdS20, (r-B,m-B), ara-14, proA2, lacY1, galK2, rpsLZ0 (Smr), nyl-5, mtl-1, supE44, X-) carrying the appropriate plasmid was inoculated into a 1-ml culture of M9 media (16), 0.5% casamino acids, 20 pg/ml tryptophan, 50 pg/rnl ampicillin and grown overnight. The culture was then diluted 10-fold into 5 ml of M9, 0.5% casamino acids, 50 pg of ampicillin and grown for 1 h at 30 "C with aeration. Twenty-five p1 of 1 mg/ml indoleacrylic acid in ethanol was added, and the cultures were grown an additional 2 h at 30 "C. The cells were then collected and solubilized in 5% SDS, 10% P-mercaptoethanol at 95 "C for 5 min. Proteins were then separated by SDS-PAGE (20). This method was also scaled up 20-fold for a large scale preparation of protein.
Subunit 6 antibodies were affinity-purified against the fusion protein as described by Olmsted (21). Following electrophoresis the SDSsolubilized proteins from the indoleacrylic acid-induced E. coli were separated by SDS-PAGE and transferred to nitrocellulose (22). The strip of the nitrocellulose that carried the fusion protein was incubated with rabbit polyclonal antibodies raised against the low molecular weight subunits of the yeast cytochrome bcl complex (23). Anti-bodies binding to the fusion protein were removed from the nitrocellulose with acid (21).
Mitochondria Preparation-Mitochondrial membranes were isolated by a modification of the method described by Needleman and Tzagoloff (24). Cells were grown overnight in 5-10 ml of liquid media or on plates. Cells were harvested, transferred to an Eppendorf tube, washed once with distilled water, and then resuspended in 400 pl of 50 mM Tris/HCI, pH 7.4, 400 mM mannitol, 2 mM EDTA. Four hundred mg of glass beads were added, and the tubes were vortexed at maximum speed for 10 min at 4 "C.
Following vortexing, an additional 400 p1 of buffer was added. The tubes were vortexed briefly and then centrifuged at 750-1000 X g for 10 min at 4 "C. The supernatants were transferred to fresh tubes and recentrifuged at 14,000 X g for 15 min to pellet mitochondrial membranes. The pellets were resuspended in 400 pl of buffer and recentrifuged. The pellets from the second centrifugation were then resuspended in 50-100 p1 of the buffer and used directly for assays or gels.
Intact mitochondria were isolated using the zymolyase method to lyse the yeast cells as previously described (25).
ATPase Assays-Mitochondrial oligomycin-sensitive ATPase was assayed by a modified method of Chan and Schneider.' Mitochondrial membranes, in a total volume of 135 pl, were preincubated in 365 p1 of 7.5 mM Tris/SO,, pH 6.0, 250 p M MgSO,, 250 p M ATP, 100 mM sucrose with or without 2.5 pg/ml oligomycin for 10 min at room temperature. Following this incubation, 125 pl of 0.2 M Tris/SO,, pH 7.0, 20 mM MgSO,, 20 mM ATP was added, and the mixture was incubated at 30 "C for 10 min. The assay was stopped by adding 1 ml of 1 M H2S04, 10% powdered activated charcoal and placed on ice. Tubes were then centrifuged at 1000 rpm for 5 min to remove the charcoal. Five hundred pl of the supernatant was placed in a fresh tube, and 500 p1 of phosphate reagent (2% ascorhic acid, 0.5% ammonium molybdate, 1.2 M HZSO,) was added. The tubes were incubated at room temperature for 90 min for color development, and the absorbance was read at 750 nm.

RESULTS
Activities of Respiratory Chain Complexes in a Strain with the Disruption qcrGdl:URA3 Allele-Mitochondrial membranes from the yeast strain carrying the qcr6al:URAS disruption were devoid of ubiquinol-cytochrome c oxidoreductase activity as shown in Fig. 1A. This differs from a deletion of the entire coding region for QCRG, where membranes still contain 50% of their wild-type cytochrome c reductase activity (9). Cytochrome c oxidase activity was diminished approximately 50% in some strains carrying the disruption (Fig. lA), but in other strains this activity was unchanged. The disruption strain had normal amounts of oligomycin-sensitive ATPase activity (results not shown).
A strain carrying the qcr6al:URAS allele was also examined and was found to resemble the qcr~al;UR~d strain in loss of ubiquinol-cytochrome c oxidoreductase activity while retaining normal levels of cytochrome c oxidase as shown in Fig.  1B.
Spectral Analysis of Cytochromes in a Strain with the Disruption qcrGdl:URA3 Allele-Mitochondrial membranes from the yeast strain carrying the qcrGal:URA3 disruption lacked the optical spectrum characteristic of the cytochrome bc, complex as shown in Fig. 2. An optical spectrum of membranes from a yeast strain carrying a wild-type QCRG allele is shown in the upper truce of Fig. 2. The spectrum shows the typical absorption at 600-605 nm due to cytochromes a and ' S. Chan and D. Schneider, unpublished data. aB, a peak at 560-562 nm due to the b cytochromes, and a shoulder at 550-553 nm due to cytochrome c1 and cytochrome An optical spectrum of membranes from a strain carrying the qcr6dl:URA3 disruption allele is shown in the bottom trace of Fig. 2. The spectrum consists of an absorption band at approximately 552 nm attributable to cytochrome c1 and cytochrome c, though due to the large reduction in the intensity of this band, it is probably mostly due to cytochrome c. The typical absorption peak at 600-605 nm attributed to cytochromes a and a3 is apparent in normal amounts. The spectrum of the qcr6dl:URAS disruption strain completely lacks the 560-562 nm absorption band due to the b cytochromes.
Because mature cytochrome b was missing in the disruption strain, mitochondrial translation products were labeled in the presence of cycloheximide and analyzed by SDS-PAGE. Translated apocytochrome b was clearly present in normal amounts (results not shown), indicating that the defect in cytochrome b is in a post-translational event.
Analysis of Nuclear Encoded Subunits in a Strain with the Disruption qcr6til:URAS Allele-Because the defect in this C. qcrGdl:URA3 FIG. 2. Cytochrome & spectra from strains with the wild-type QCRS and the disruption qcr6al:URAS alleles. Mitochondrial membranes were suspended in 50% glycerol with 1% Triton X-100 and scanned from 520 to 620 nm in an Aminco DW-2A dual wavelength spectrophotometer. Dithionite-reduced minus ferricyanideoxidized difference spectra were calculated using a digital oscilloscope (23).
mutant is localized to the mitochondrial cytochrome bcl complex, we examined the nuclear gene products that were present in mitochondrial membranes of a strain carrying this disruption allele, using antibodies against the nuclear encoded subunits of the complex (23). Antibodies against subunit 6 proved difficult to obtain, and, probably due to its small size and predicted solubility, it is rapidly cleared from immunized animals. We did possess a large amount of rabbit polyclonal sera, initially raised against the low molecular weight subunits of the yeast cytochrome bcl complex that cross-reacted with subunit 6 in purified bc, complex. However, this sera also cross-reacted with innumerable yeast proteins. To alleviate this problem, the gene for subunit 6 was fused in frame to the E. coli TrpE gene. This allowed for the induced production of large amounts of subunit 6 antigen in E. coli, where crossreacting yeast proteins would be absent. This E. coli-produced subunit 6 antigen was used for affinity purification of highly specific subunit 6 antibodies from rabbit polyclonal antibodies raised against the low molecular weight subunits of the yeast cytochrome bcl complex (23).
Both mitochondrial membranes and whole mitochondria were probed with the subunit-specific antibodies seen in Fig.  3. Identical results were seen with both protein sources. The membranes from strains carrying either a wild-type QCR6 allele or a deletion qcr6Al:LEUZ allele (9) contained similar amounts of all the subunits probed for, except of course for subunit 6, which was absent in the deletion strain (Fig. 3). The strain with the disruption qcr6dl:URA3 allele, despite overloading with protein, contained normal amounts of only core protein 1, while all of the other subunits were absent or present in amounts that were significantly reduced. These include subunit 6, cytochrome cl, the Rieske iron-sulfur protein, and subunit 7 (not shown). This pattern of subunit loss is similar to that seen in either a subunit 8 null mutant, a cytochrome c1 null mutant, or a cytochrome b null mutant The subunit 6 antibodies are from rabbit polyclonals against the low molecular weight subunits of the yeast bcl complex (23) that were affinity-purified against 7'rpE:QCRG fusion protein. The control is an antibody to a cytochrome c oxidase subunit to show overloading of the qcr6al:URAS lane.
(11) and is characteristic. of a loss of assembly of the complex.
Complementation of the qcr682:URAS Disruption Strain with a High Copy Library-The availability of a stable mutant of QCRS (qcrGdB:URA3) having a petite phenotype allowed us to learn more about the function of this subunit by looking for extragenic suppressors that could complement the qcr6a2:URA3 petite phenotype when present in high copy (29). We thus attempted to complement the qcrGa2:URA3 strain with a high copy 2 p LEU2-based yeast genomic library. Because the qcr6a2:URA3 allele was known to complement with a wild-type copy of QCR6, this experiment had an internal control. Clones carrying QCR6 should be identified using this approach.
The strain MES20, which contains a qcr6a2:URA3 allele, was transformed with a high copy yeast genomic library. Yeast transformed with the plasmid-borne library were first selected on defined medium lacking leucine. About 10,000 Leu+ transformants were then replica-plated to YPG (1% yeast extract, 2% peptone, 3% glycerol) plates a t 37 "C. After 5 days of growth, colonies that grew were picked and plated onto media with neither leucine nor uracil. Those that grew on both plates were saved as containing potential suppressors. Those that were uracil auxotrophs were considered intragenic revertants and were discarded.
The potential suppressors were then tested for plasmid linkage to growth at 37 "C on nonfermentable carbon sources. These strains were streaked onto 1% yeast extract, 2% peptone, 10% dextrose plates and grown a t 30 "C. Under these conditions there should be little selection for respiratory competence, and the unstable library plasmid should be spontaneously lost. Colonies from these plates were then tested on defined medium minus leucine and on YPG plates a t 37 "C.
If the suppression is linked to the library plasmid, then when the strain becomes a leucine auxotroph it should also become a temperature-sensitive petite. When the strain retains leucine prototrophy, it should continue to be a suppressor. This was the case for seven different strains. No derived colonies of these strains were found to be Leu+ pet-or leu-Pet+, as would be expected from a chromosomal suppressor.
The high copy library plasmids were rescued from the seven suppressed strains and transformed into E. coli for amplification. T o identify suppressor plasmids that might carry a wild-type QCRG, the plasmids were digested with either AccI or SphI, separated by agarose gel electrophoresis, transferred to nylon, and probed with a 715-bp AccI fra, ument encompassing QCR6. Three of the clones hybridized with the QCRG probe and gave restriction fragments of the expected size for the QCRG gene. However, clones 16, 20, 22, and 24 failed to hybridize with the QCRG probe.
Restriction Mapping and Characterization of the Extragenic Complementing Plasmids-The clones 16,20,22, and 24 all suppressed the qcrGa2:URA3 allele but were extragenic to QCRG. These clones were subjected to restriction analysis and mapped. All four clones were found to have overlapping maps that stretched over 9 kb of the yeast genome as shown in Fig.  4. By eliminating nonoverlapping regions of the clones, the complementing region was confined to 2.8 kb.
The restriction map of this region was suspiciously similar to the restriction map of a gene previously cloned in this laboratory (30). This gene, QCR9, codes for subunit 9, the 7.2-kDa subunit, of the yeast mitochondrial cytochrome bc, complex.
These overlapping clones were digested with either XbaI or EcoRI and PstI, subjected to Southern analysis, and hybridized with a 1.9-kb fragment encompassing the gene for QCRS. All of these overlapping clones hybridized with the QCR9 probe, and they all gave fragments of the expected size for the QCRS gene. This 2.8-kb fragment includes all of the elements required for expression of QCRS (30).
Partial Restoration of Cytochrome c Reductase Activity in the Complemented Strains-To identify the effect overproducing QCRS has on the respiratory chain in a qcr6a2:URA3 strain, ubiquinol-cytochrome c oxidoreductase and cytochrome c oxidase activities were measured. These activities were measured in mitochondria from the strains overproducing either QCR6 or QCRS in a qcrGd2:URA3 background and compared with those from the wild-type parent strain and a qcrGa2:URA3 strain. Respiratory chain activities in these strains are shown in Fig. 1B. As can be seen, the high copy QCR6 restores the cytochrome c reductase activity up to 30% -

22
" " " " " " " " " " " " " " of that in the wild-type parent, while the high copy QCRS restores the activity to about 10% that of the wild type.

DISCUSSION
We previously described a novel mutation of QCR6 that causes a temperature-sensitive petite phenotype in S. cerevisiae. In this paper mitochondria from the petite strain carrying the qcr6al:URA3 disruption allele were biochemically characterized, and suppressors of this petite strain were selected, identified, and characterized.
Several findings indicate that the petite phenotype is due to a lack of cytochrome bcl complex. Mitochondria from the strain carrying the disruption qcr6al:URAS allele were devoid of ubiquinol-cytochrome c oxidoreductase activity but had normal amounts of cytochrome c oxidase and oligomycinsensitive ATPase. Optical spectra of mitochondrial membranes from the petite strain point to a loss of both cytochromes b and cl, while cytochromes a and a3 are present. This loss of cytochrome bcl complex is similar to what is seen as a result of mutations in either subunit 7, subunit 8, or cytochrome c1 (11).
Immunological probing for subunits in the petite strain indicated that most of the subunits of the bcl complex are missing. Most of the nuclear encoded subunits of the cytochrome bcl complex are known to be rapidly degraded in the absence of complete assembly of the complex (31). The apparently toxic hybrid protein produced from the qcr6al:URA3 allele was undetectable with the subunit 6 antibodies used in this study. This may be due to a lack of cross-reactivity with the poisonous protein or the presence of the toxic protein at levels below detection. The latter possibility is consistent with the low levels of transcript from the locus responsible for the petite phenotype (data not shown).
On the basis of these findings we suggest that the deleterious derivative of subunit 6 disrupts assembly of the cytochrome bc, complex. Since only a small number of bcl complexes is undergoing assembly at one time, a low level of toxic protein could cause such a deleterious effect. Our results are consistent with an assembly pathway as outlined in Fig. 5. In such a pathway the aberrant subunit 6 may bind to cytochrome c1 and cause it to become unstable. This would remove cytochrome c1 from the assembly pathway and destabilize the residual subcomplex. This is consistent with biochemical evidence that subunit 6 and cytochrome c1 form a subcomplex during fractionation of purified cytochrome bcl complex (32) and likewise with genetic evidence that cytochrome c1 is unstable in a yeast strain that is rhoand disrupted for QCR6 (10).
Extragenic high copy suppressors of the disruption qcr682:URA3 allele were found by complementation and identified as QCR9, the nuclear gene that encodes subunit 9 of the cytochrome bcl complex (30). This constitutes genetic evidence of an interaction between these two subunits and agrees with biochemical findings that these two subunits form a subcomplex with cytochrome c1 (33,34).
The phenotype of the disruption qcrGa2:URA3 allele was also suppressed by a wild-type copy of QCR6. Suppression by high copy expression of QCR6 was expected, since the petite phenotype of the qcr6al:URA3 strain was suppressed by mating of a rho' strain carrying a wild-type copy of QCR6. Suppression by QCR6 differs from that by QCRS in that the former requires only a single copy of the gene, as evidenced by the phenotype of a qcr6al:URAd X QCR6 diploid, while multicopy expression of QCRS above chromosomal copy levels was required to suppress the petite phenotype of the qcr6a2:URA3 strain. and co-workers (35) and Crivellone and co-workers (11) with findings presented here. In the model, core protein 1 ( I ) and core protein 2 (ZZ) form a stable ( S ) subcomplex, while cytochrome b (b) subunits 8 ( 8 ) and 7 (7) form a subcomplex that is believed to be unstable ( U ) . The core protein subcomplex and cytochrome b-subunit 7subunit 8 subcomplexes then associate to form a subcomplex with intermediate stability ( S / U ) . A separate nucleation site originates from subunits 6 (6) and 9 (9), which combine and then associate with cytochrome el (el) into a subcomplex of intermediate stability ( S / U ) . The cl subcomplex then associates with the b-core subcomplex to form a stable ( S ) cytochrome bel complex without iron-sulfur protein (ZSP). Insertion of the iron-sulfur protein then completes the assembly pathway (11). We propose that the aberrant form of subunit 6 (6*) combines with cytochrome el before subunit 9, causing the c1 to be degraded by protease, thus preventing completion of the assembly pathway. This can be suppressed by overproducing subunit 9, which binds to the aberrant subunit 6 and sequesters it from the pathway. A normal subunit 6, either in single or multiple copies, forms a subunit 6-9 dimer, which competes with the aberrant subunit 6 for cytochrome el.
Suppression of the qcr6a2:URA3 petite phenotype by either QCRS or QCR6 leads to only a partial recovery of cytochrome c reductase activity, but this proved to be enough to permit growth on nonfermentable carbon sources. Schoppink and coworkers (35) previously showed that as little as 5% of the normal ubiquinol-cytochrome c oxidoreductase activity is sufficient for yeast to grow on nonfermentable carbon sources, although one might expect this activity threshold to vary among strains.
An alternative explanation for the lack of bcl complex is that the aberrant subunit 6 removes subunit 9 from the assembly pathway, since deletion of QCRS also results in a temperature-sensitive petite phenotype (30). We feel this explanation is unlikely, since deletion of QCRS leads to a different assembly phenotype (30). However, we cannot exclude the possibility that loss of both subunit 6 and subunit 9 may lead to a phenotype similar to the qcr682:URA3 disruption allele.
Since overexpression of QCRS protects against the effects of the aberrant subunit 6, we suggest that subunits 6 and 9 form a subcomplex before they bind to cytochrome c1 (Fig. 5). Perhaps the toxic derivative of subunit 6 binds better to cytochrome c1 than to subunit 9, leading to rapid degradation of cytochrome cl. Overexpression of subunit 9 may override the otherwise weak interaction with subunit 6 and thus cause the poisonous subunit 6 to be removed from the pathway. Since subunit 6 is not required for assembly of the bcl complex (9), the complex is assembled without it.
The normal subunit 6, either in single or multiple copies, permits the formation of a subunit 6-subunit 9 dimer, which competes with the aberrant subunit 6 for cytochrome cl. The fact that a normal subunit 6 rescues only 30% of the bcl complexes when expressed at high copy number may simply reflect a lower affinity of the subunit 6-subunit 9 dimer than of the mutant subunit 6 for the binding site on cytochrome C1.