Introduction of the Chloroplast Redox Regulatory Region in the Yeast ATP Synthase Impairs Cytochrome c Oxidase*

The ATP synthase is under a number of mechanisms of regulation. The chloroplast ATPase has a unique mode of regulation in which activity is controlled by the redox state in the organelle. This mode of regulation is determined by a small unique region within the γ-subunit and this region contains two cysteine residues. Introduction of this region within the yeast γ-subunit causes a defect in oxidative phosphorylation. Oxidative phosphorylation is restored if the cysteine residues are replaced with serine. Biochemical analysis of the chimeric mitochondrial ATPase indicates that the ATP synthase is not largely altered with the cysteine residues in either the oxidized or reduced states. However, the level and activity of cytochrome c oxidase are decreased by about 90%, whereas that of NADH dehydrogenase and cytochrome c reductase are unchanged as compared with the wild-type enzymes. The level and activity of cytochrome c oxidase are restored with replacement of the cysteine residues with serine in the regulatory region. These results indicate that the chimeric ATP synthase containing cysteine, but not serine, decreases the expression or assembly of cytochrome c oxidase with little effect on the activity of the ATP synthase.

wasteful loss of ATP under conditions where ATP is not being made (7). The inhibitor protein is not present in bacteria, but analogous regulation is thought to be effected by the C-terminal of the ⑀-subunit (8 -10). (The bacterial ⑀-subunit is homologous to the mitochondrial ␦-subunit.) The ␥-subunit of the chloroplast ATPase contains a unique disulfide bond that inhibits ATP hydrolysis by CF1, but in the reduced form, is non-inhibitory (11). This regulatory region thus acts as a redox sensor to inhibit the chloroplast enzyme under conditions where ATP is not being made and may act in concert with the inhibitory action of the C-terminal domain of the ⑀-subunit (12).
Two cysteine residues, Cys 199 and Cys 205 , in the chloroplast ␥-subunit are responsible for the redox regulation of the chloroplast ATP synthase (11). The region containing Cys 199 and Cys 205 is unique to the chloroplast ␥-subunit. However, introduction of this region into the bacterial enzyme has been shown to convey the redox regulation to the bacterial enzyme (13).
There are five enzyme complexes that participate in oxidative phosphorylation: complexes I, II, III, IV, and V corresponding to NADH dehydrogenase, succinate dehydrogenase, cytochrome c reductase, cytochrome c oxidase, and the ATP synthase, respectively. In yeast, supercomplexes have been identified that are composed of Complexes III and IV (14,15). In mammalian cells, almost all of Complex I is in a supercomplex with Complexes III and IV. Furthermore, the ATP synthase has been reported to be in a supercomplex with the phosphate carrier and ATP/ADP translocase (14 -18). These supercomplexes are thought to add an additional level of efficiency to the reaction pathway. In addition, in yeast and mammalian cells, the ATP synthase forms a dimer (14), and in yeast, it is at least partly responsible for formation of cristae (19). The ATP synthase dimer is mediated by subunit g of the ATP synthase. Deletion of subunit g eliminates the dimer form of the ATP synthase and also eliminates the cristae in the yeast mitochondrion suggesting a role of the ATP synthase in the formation of cristae (19). Deletion of subunit g also has an effect on the activity of cytochrome c oxidase suggesting the importance of the dimer form of the ATP synthase in expression or stability of cytochrome c oxidase (20).
We have investigated the structure function relationship of the redox regulatory region of the chloroplast ATP synthase by making chimeric constructs of the yeast and chloroplast ␥-subunit. By use of homologous recombination in yeast, five chimeric constructs have been made to test the capacity of regions within the chloroplast ␥-subunit to confer redox regulation to the yeast ATPase. The chimeric construct containing the entire putative regulatory region of the chloroplast ␥-subunit, when inserted in the yeast ␥-subunit, inactivated oxidative phosphorylation in the yeast, as judged by biochemical assays and the ability of the yeast to grow on a non-fermentable carbon source. However, the defect was due to the nearly complete loss of cytochrome c oxidase activity, with only mild alterations in the ATP synthase or in the other complexes of the electron transport chain. The results of this study provide new structure/ function information on the chloroplast regulatory region and suggest that the ATP synthase is either directly or indirectly related to the function of cytochrome c oxidase.
Construction of Yeast/Chloroplast Chimeric ␥-Subunits-Yeast/chloroplast chimeric ␥-subunit were made using gap repair (22). The region of the gene coding for the regulatory region of the chloroplast ␥ subunit was amplified by polymerase chain reaction (PCR). The 5Ј ends of the PCR primers contained 30 bases that corresponded to the target point for homologous recombination. The PCR products were co-transformed with a linear plasmid DNA (pYATP3NcoI) containing yeast ATP3 into W303-1A (23). The plasmid DNA was made linear by making a single restriction cut within ATP3. Upon transformation into yeast, homologous recombination occurs between the PCR product and the corresponding linear plasmid. This recombination event replaced the corresponding domain in ATP3 with the regulatory region from chloroplast enzyme and circularized the plasmid DNA. The recombinants were selected by growth of cells on minimum media devoid of tryptophan. The plasmids were extracted from the transformants and transformed into Escherichia coli XL1-Blue by electroporation as detailed in the instrument manual (Bio-Rad). The plasmid DNA was purified from E. coli and sequenced (University of Chicago Cancer Research DNA Sequencing Facility). Supplemental Table S2 shows a list of plasmids used in this study.
The concentration of the soluble F 1 -ATPase was determined by the Bradford (26) method. The concentration of F 1 F 0 -ATP synthase was determined by densitometry of the Coomassie Blue staining intensity of the ␣and ␤-subunits after separation by SDS-gel electrophoresis using purified F 1 -ATPase as a standard.
Biochemical Studies-Mitochondria were isolated from yeast strains as described (27). The concentration of mitochondrial protein was determined using the BCA protein assay (26). Western blot analysis (28) was performed using rabbit antibodies directed against yeast Cox2p, Cox3p, cytochrome b, Rieske FeS protein (Gifts from Dr. Rosemary Stuart, Marquette University, Milwaukee, WI), and chicken antibodies against ␣and ␤-subunits of yeast F 1 .
Mitochondrial respiratory enzyme activities (NADH dehydrogenase, cytochrome c reductase, and cytochrome c oxidase) were measured as described (29 -31). ATPase activity was determined by the coupled enzyme reaction (27) at 30°C. As indicated, the samples were incubated with 10 mM dithiothreitol (DTT) 3 or 100 M CuCl 2 for 30 min before the assay.
ATPase activity of F 1 F 0 -ATP synthase was measured after reconstituting the enzyme into liposomes as described (32) with some modifications. ␣-Phosphatidylcholine (60 mg) was suspended in 2 ml of REV buffer (50 mM KCl, 0.2 mM EDTA, 20 mM TrisSO 4 , pH 8.0). The lipid dispersion was placed in a 10-ml glass tube and sonicated for 3 min at 10°C (model G112SP1G, Laboratory Supplies Co., Hicksville, NY). Reconstitution of the ATP synthase in liposomes was performed by mixing 0.3 ml of the liposomes (30 mg/ml), 0.15 ml of REV buffer, and F 1 F 0 (90 g) to a final volume of 0.54 ml. Triton X-100 (60 l of a 10% solution) was added to the mixture with vigorous mixing. The mixture was stirred slowly for 10 min. after which Bio-Beads (200 mg) were added and the mixture was stirred for 3 h. The slightly turbid proteoliposomes were carefully removed avoiding the Bio-Beads. The proteoliposomes were stored at 4°C.
Enzyme Kinetics-For determination of K m , the ATPase activities were measured in the presence of MgATP ranging from 2 M to 0.8 mM. Kinetics parameters were determined according to simple Michaelis-Menten kinetics using Enzyme Kinetics Module from Sigmaplot 9.0 (Systat Software Inc., San Jose, CA).
Cytochrome Spectrum Analysis-Submitochondrial particles (submitochondrial particles (2.5 ml at 20 mg/ml) in 0.25 M sucrose, 20 mM Tris-Cl, pH 7.5) were dissolved with addition of KCl (500 mg), 1 M Tris, pH 7.5 (0.5 ml), and 10% potassium deoxycholate (1 ml), while mixing. The volume was adjusted to 10 ml with distilled water. The solubilized submitochondrial particles were centrifuged at 125,000 ϫ g for 15 min and potassium cholate was added to the supernatant to 1%. The hemes were oxidized with potassium ferrycyanide (0.015%) or reduced with sodium dithionite. Absorbance difference spectra were recorded at room temperature using a HP8453 diode array spectrophotometer.
Association of Cytochrome c Oxidase and the ATP Synthase-The association between the yeast ATP synthase and cytochrome c oxidase was studied by co-purification of cytochrome c oxidase with ATP synthase. ATP synthase was purified from mitochondria isolated from a strain (HSY202) in which the ␤-subunit had a His 6 tag on the NH 2 terminus. Mitochondria (10 mg/ml) were adjusted to 1.5% digitonin, the solution was centrifuged at 150,000 ϫ g for 15 min at 4°C, and the supernatant was applied to a nickel-Sepharose column (5 ml, GE Healthcare) equilibrated with buffer composed of 10% glycerol, 0.3 M NaCl, 50 mM phosphate, 5 mM ⑀-aminocaproic acid, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 30 mM imidazole, and 0.01% digitonin, pH 7.5. The protein was eluted with the wash buffer containing 300 mM imidazole. Fractions containing the eluted portion were pooled and analyzed using Western blot with antibodies indicated.

RESULTS
The primary structure comparison between yeast and chloroplast ␥-subunit is shown in Fig. 1A. Overall the subunits share 34% identical amino acids and 46% homology. This is similar to the percent identity and homology between ␥-subunits from yeast and bovine (42 and 54%, respectively). The central region, residues 174 -232 in the chloroplast ␥-subunit, contains regions that are distinct from those in the yeast ␥-subunit and within that region reside the cysteine residues required for redox regulation, Cys 199 and Cys 205 . This is the only region identified by analysis of the primary sequence that obviously differed between the yeast and chloroplast ␥-subunit, suggesting that no other regions are required for redox regulation. The putative redox regulatory region was divided into three regions, I, II, and III, colored in Fig. 1A as blue, green, and red, respectively. These regions are highlighted in the model of the structure of the yeast F 1 -ATPase with the corresponding colors for regions I and III (blue and red, Fig. 1, B and C). Region II (green) does not have any corresponding residues in the yeast ␥-subunit and is thus not present in the model. To test the importance of each of these regions in the redox regulation, four chimeric constructs were made in which the regions of the chloroplast ␥-subunit were inserted at the corresponding regions of the yeast ␥-subunit. These four chimeric constructs correspond to insertion or replacement of regions: (II), (I, II), (II, III), and (I, II, III).
The four constructs were made in yeast using homologous recombination, which allowed the precise insertion of these regions within the gene. The chimeric gene constructs were transformed into a yeast strain devoid of the genes encoding the ␥-subunit (ATP3) and that encoding the ␤-subunit (ATP2). The resulting strains were transformed with the gene encoding a His 6 tagged derivative of the ATP2 gene. The atp2 Ϫ mutation was necessary because yeast with an atp3 Ϫ mutation rapidly become petite (loss or deletion of the mitochondrial DNA), which prevents biochemical analysis of the ATP synthase.  (1-5) are shown for the yeast F 1 -ATPase. B and C, partial model of the yeast F 1 -ATPase showing subunits, ␤ E , ␣ TP , ␥, ␦, and ⑀. The regions corresponding to I and III are colored in blue and red, respectively. Note: the region corresponding to yeast residues ␥61-73, is not present in the model, but is shown in C as a yellow loop for reference. The images were made with PyMOL (44).
However, the percentage of petites formed is dramatically reduced in the atp2 Ϫ , atp3 Ϫ double mutant strain and low in the atp2 Ϫ mutant strain (33). The His 6 derivative of ATP2 also allows rapid purification of the F 1 -ATPase or the F 1 F 0 -ATPase for biochemical studies. Supplemental Table S1 shows a list of all of the strains used in this study. Fig. 2 shows the growth of the yeast cells on complete medium containing glycerol as the carbon source (YPG) or minimal medium with glucose as the carbon source (synthetic media). Cells defective in oxidative phosphorylation are unable to grow on YPG medium, but can grow on medium with glucose as a carbon source. The results indicate that insertion of region (II) alone did not alter the cells' ability for growth on YPG medium, whereas insertion of regions (I, II) had a modest negative affect, insertion of regions (II, III) had a moderate affect, and insertion of regions (I, II, III) eliminated the ability of the cell to grow on YPG medium. To determine whether the Cys residues in region II were responsible for the defect in chimera (I, II, III), these residues were mutated to Ser and tested in the same manner. Replacement of Cys with Ser in the largest chimeric construct (I, II, III) (C225S,C231S) restored the ability of the cell to grow on YPG. This result indicates that the insertion of regions (I, II, III), but not any of the smaller inserts, inactivated oxidative phosphorylation in the yeast strain. Furthermore, cysteine residues at the positions that correspond to chloroplast Cys 199 and Cys 205 , are required for the inactivation of oxidative phosphorylation.
The ATPase activity was measured for the wild-type and mutant mitochondrial F 1 -ATPase in the isolated mitochondria, the chloroform released enzyme, purified F 1 , and in some cases, the F 1 F 0 -ATP synthase ( Table 1). The results indicate that the total activity was reduced by about 60% for the yeast strain containing (I, II, III), with a corresponding decrease in oligomycin sensitivity. The F 1 -ATPase is selectively released with chloroform extraction and releases only assembled enzyme (34). The total activity of the chloroform-released enzyme from chimera (I, II, III) mirrored the activity of the isolated mitochon-dria supporting the conclusion that the ATPase activity is from the F 1 -ATPase. In contrast, the level of the ATPase activity from the mitochondria isolated from the remaining mutant strains, and the corresponding chloroform-released enzyme, were not decreased by more than 25% compared with the wildtype strain. The mitochondrial enzyme showed a corresponding small decrease in oligomycin sensitivity. Surprisingly, in comparison to the wild-type enzyme, oxidation with CuCl 2 did not largely inhibit the ATPase activity relative to reduction with DTT. Separate experiments using purified F 1 -ATPase from the HSY205 indicated that disulfide bond formation occurred readily between the cysteines in the regulatory region (not shown).
The F 1 and F 1 F 0 -ATPase were purified from the strains to determine whether the specific activities of the enzymes were altered with the insertion of the chloroplast regions ( Table 1). The ATPase activity from the F 1 purified from the chimera strain (I, II, III) did not decrease by more than 20% as compared with the wild-type enzyme. The specific ATPase activity of F 1 F 0 isolated from chimera (I, II, III) was not lower than that of either the wild-type or the C2S chimera strain (HSY204C2S). The largest alteration was in the K m for ATP of the chimeric F 1 F 0 -ATP synthase. It was about twice that for the (I, II, III) mutant enzyme (105 M) as compared with that of the wild-type strain (59 M) (when measured under oxidizing conditions) and this difference was eliminated after reduction with DTT. Thus, based on these studies, the activities of F 1 and F 1 F 0 were not largely altered by the insertion of the chloroplast regulatory region (I, II, III).
Selective Decrease in Cytochrome c Oxidase Activity-Prior studies have indicated that the activity of the ATP synthase must be decreased by more than 85% before there is a negative growth phenotype on YPG medium (35). The biochemical studies of the chimeric yeast/chloroplast enzyme (I, II, III) indicated that the activity of this enzyme was not nearly sufficiently inhibited to be responsible for the negative growth phenotype. Thus, studies were performed to determine whether there were defects in any of the enzymes of the electron transport chain, which may be responsible for the inability of the yeast to grow on medium containing a nonfermentable carbon source. Specific activities were determined for NADH dehydrogenase, cytochrome c reductase, and cytochrome c oxidase (Table 2). FIGURE 2. Growth phenotype of the yeast strains with the chimeric constructs. The growth of the cells are shown on solid plates containing minimal medium (synthetic media, SD) and complete medium containing glycerol as the carbon source (YPG). The cells were added to water at three dilutions, as indicated, and grown at 30°C. The negative control is HSY201 transformed with vectors pRS304 and pRS306.

TABLE 1 ATPase activities of the chimeric enzymes
The specific ATPase activity of the wild-type (WT) and the chimeric enzymes is shown in the table as purified in mitochondria (3rd column), the chloroform released enzyme (5th column), purified F 1 -ATPase (6th column), purified F 1 F 0 -ATPase (7th column), and the percentage oligomycin sensitive ATPase of the purified mitochondria (4th column). The percentage inhibition (3rd and 5th columns in brackets) is calculated based on rates in the presence of DTT and CuCl 2 . The K m for ATP of F 1 F 0 -ATPase is shown in the 8th column. The K m of ATP for the chimeric enzyme was measured both in reduced state (DTT) and oxidized state (CuCl 2 , in brackets). The results are an average of at least 2 separate preparations and multiple assays. Supplemental Fig. 1S shows analysis of the purified F 1 -ATPase and F 1 F 0 preparations by SDS-polyacrylamide gel electrophoresis. NA, not applicable; ND, not determined. The results indicate that cytochrome c oxidase activity is decreased by nearly 95% in the HSY204 strain (I, II, III), but activity is decreased by just 35% when the Cys 225 and Cys 231 are replaced with Ser (HSY204C2S). Studies were performed to determine whether the decrease in cytochrome c oxidase activity was due to an inhibition of the activity leaving an assembled and stable complex or if the cytochrome c oxidase is absent from the mitochondria. Fig. 3A shows difference spectra of mitochondria isolated from the corresponding wild-type and mutant strains. The spectra indicate that heme aa 3 is largely reduced in the HSY204 strain containing the regulatory region (I, II, III), at near normal levels in the HSY204C2S strain. In contrast, cytochromes b, c, and c 1 were at normal levels in the HSY204 strain (I, II, III). Fig. 3B shows the Western blot analysis used to determine whether the subunits of cytochrome c oxidase are present in the HSY204 strain (I, II, III). The results indicate that Cox2p and Cox3p were both decreased but the percentage decrease was not consistent with the observed decrease in cytochrome c oxidase activity. The Western blot also demonstrates that the level of the ␣and ␤-subunits of the ATPase are decreased in the HSY204 strain (I, II, III), but not by more than about 50%. Analysis of the level of heme and activity of cytochrome c oxidase were measured from mitochondria isolated from strains HSY203, HSY205, and HSY206, and the levels were consistent with their growth phenotype on YPG medium (supplemental Fig. 2S).

Strain
Association of Cytochrome c Oxidase with the ATP Synthase-The results suggest that the ATP synthase is either directly or indirectly associated with cytochrome c oxidase. To determine whether there is a direct association of cytochrome c oxidase with the ATP synthase, the ATP synthase was partially purified by nickel-affinity chromatography (using the His 6 tag on the ␤-subunit) and analyzed for copurification of cytochrome c oxidase (Fig. 4). The results indicate that Cox2p and Cox3p of cytochrome c oxidase co-purified with the ATP synthase suggesting a direct interaction of cytochrome c oxidase with the ATP synthase. The control indicated that cytochrome c oxidase did not bind to the nickel-Sepharose column in the absence of the His 6 tag on the ATP synthase. However, the association of ATP synthase and cytochrome c oxidase is not strong, as it is not observed when the mitochondrial membrane is treated with 1% dodecyl-␤-D-maltoside (data not shown).
Model of the Chloroplast Regulatory Loop-The conformation of region (I, II, III) within the context of the yeast ␥-subunit and F 1 -ATPase was homology modeled using Molecular Operating Environment (MOE 2007.09, Chemical Computing Group, Inc., Montreal, Canada). In addition, a loop in the ␥-subunit (residues 60 -70) was modeled with MOE because this region is missing in the yeast model and the corresponding residues are also missing in the bovine model. The resulting model shown in Fig. 5 provides a framework within which to interpret the results. The regulatory region is on the face opposite the ␦and ⑀-subunits. The model shows that the regulatory region is capable of interacting with ␤ E , ␤ TP , and ␣ TP . The model indicates that potentially, the regulatory region can interact with the catalytic and non-catalytic subunits of the ATPase and has the potential to insert into the interface between ␤ E and ␣ TP . The model places the two regulatory Cys residues at the end of a loop. This loop structure is similar to the redox loop in the periplasmic domain of the transmembrane electron transporter, DsbD (Protein Data Bank 1JPE) (36). Like the chloroplast regulatory region, this redox loop undergoes oxidation/reduction of two cysteine residues, which are spaced FIGURE 3. Selective loss of cytochrome c oxidase. A, difference absorbance spectra (reduced minus oxidized) of detergent-dissolved mitochondrial protein. B, Western blot analysis was performed with mitochondrial proteins isolated from the strains and antibodies, as indicated, as described under "Materials and Methods."

TABLE 2 Activity measurements of respiratory enzyme complexes
The enzyme activities of NADH dehydrogenase (micromole of Fe(CN) 6 /min/mg of protein), cytochrome c reductase (micromole of cytochrome c/min/mg of protein), and cytochrome c oxidase (nanomole of O/min/mg of protein) were measured as described under "Materials and Methods." The results are an average of at least 2 separate preparations and multiple assays.  The mitochondrial ATPase was dissolved from a mitochondrial sample using 1.5% digitonin, the solution was centrifuged at 150,000 ϫ g, and the supernatant (Ni-Load) was placed on a nickel-Sepharose column. The column was washed and the ATP synthase was eluted (Ni-elute) with buffer containing 300 mM imidazole. The proteins from fractions (10 g) were separated by SDSpolyacrylamide gel electrophoresis (11%), transferred to nitrocellulose, and detected with antibodies against subunits 2 and 3 (Cox2p and Cox3p) of cytochrome c oxidase and with antibodies directed against the ␣and ␤-subunits of the yeast ATPase. As a control, the experiment was repeated with a mitochondrial preparation from wild-type yeast strain that did not have a His 6 tag on the ␤-subunit of the ATPase. The samples were treated and analyzed in the same manner as those above.

Strain
similarly (residues 103 and 109 in DsbD). This model thus provides an explanation as to how this region might interact with the F 1 , altering the catalysis, and it explains potential specific requirements for interaction with the ␣and ␤-subunits.

DISCUSSION
There are two key advances resulting from this study. First, the results of this study indicate that the regulatory region with the ␥-subunit of the ATP synthase is necessary, but not sufficient, for the redox regulation of the ATP synthase. In addition, the proposed model of this region, within the context of the structure of the yeast F 1 -ATPase, suggests an allosteric model for the regulatory role of this region. Second, the inactivation of cytochrome c oxidase by the introduction of this chloroplast regulatory region into the yeast ATP synthase suggests a direct interaction of the yeast ATP synthase in the assembly or function of cytochrome c oxidase.
Prior studies suggested that this region is sufficient to confer redox regulation. Addition of the regulatory region into the ␥-subunit of the F 1 -ATPase from cyanobacteria (37) and thermophylic bacteria (13) confers redox regulation. These studies suggested that the regulatory region was sufficient to confer redox regulation. However, the results of the chimeric enzyme made with the yeast enzyme indicate that more is required to confer redox regulation of the enzyme. Although the introduction of the cysteine containing regions, but not corresponding constructs containing serine, resulted in loss of oxidative phosphorylation in vivo, there was no demonstrable loss in the level or activity of the ATP synthase. Instead, as will be discussed later, the chimeric construct dramatically impaired cytochrome c oxidase. Introduction of the regulatory region into the ␥-subunit had no dramatic effect on a number of biochemical parameters including the kinetics of ATP hydrolysis (Table 1), coupling as determined by oligomycin sensitivity (Table 1), and stability as measured by urea denaturation studies (data not shown). In addition, there was little effect on the enzyme activities and stability when compared between the oxidized and reduced forms. Thus, despite the results from the prior studies, the introduction of the chloroplast regulatory region in the yeast ␥-subunit is not sufficient for the redox regulation of the enzyme.
The model of the structure of the redox regulatory region in the context of the structure of the yeast F 1 -ATPase does suggest a number of possible regulatory mechanisms. The chloroplast regulatory region is within an antiparallel ␤-sheet topology formed by ␤-sheets 4 and 5 of the ␥-subunit (Fig. 1). This sheet-4-loop-sheet-5 structure abuts against the C-terminal ␣-helix of the ␥-subunit and may be important for the curvature of the coiled-coil structure formed by the N-and C-terminal ␣-helices. The curvature of the coiled-coil structure is in turn critical for the structure and function of the ATP synthase as the rotation within the core of the F 1 is responsible for the changes in the conformation of the active sites during ATP synthesis and hydrolysis. The oxidation of the regulatory region may disrupt this ␤-sheet topology, thereby disrupting the coiled-coil structure and thus the structure of the active sites. Structural studies on the redox-regulated fructose-1,6-bisphosphate phosphatase suggests a similar mechanism where reduction of the disulfide bond releases the ␤1-␤2 strands of an antiparallel sheet (38).
The model shown in Fig. 5 and supplemental Movie S1 suggests that the regulatory region is close to the catalytic interface formed by ␣ TP and ␤ TP . Thus, a second possible mechanism is that the regulatory loop moves into the interface and prevents the proper conformational changes during the catalytic cycle. This mechanism is similar to that proposed for the inhibitor protein that has been shown to bind to the catalytic ␤ E interface of the bovine ATPase (39).
The model and mechanism proposed here differ considerably from an earlier model (12,40). The prior model of the chloroplast regulatory region places it at the bottom of F 1 , far from the coiled-coil region of the ␥-subunit. This prior model is dramatically different from the structure of other known structures of the F 1 -ATPase, including bovine, liver, and yeast mitochondrial, E. coli, and TF1. The prior model obviates any importance of the antiparallel ␤-sheet formed by sheets 4 and 5 and places a large structure at the base of the F 1 -ATPase. Any large structure placed at the base of F 1 , would likely impair The region corresponding to yeast residues ␥61-73 is not shown in the yeast structure, but boundary atoms of this region, residues ␥60 and ␥74, are shown as spheres to mark the border. This model is shown in the supplemental Movie S1. B, stereo image (walleyed) of the regulatory region modeled into the yeast structure. The structure is shown here as a schematic, as opposed to the line model in A. The region of residues ␥60 and ␥74 is modeled here as an ␣-helix, although there is no experimental evidence for the structure of this region. As in A, the atoms of chloroplast ␥ Cys 199 and Cys 205 are shown as spheres.
interaction with the F 0 components and rotation of the central stalk making this location unlikely. Finally, there are no easy explanations as to how redox regulation could occur if this regulatory element was so distant from either the central stalk or the active site of the enzyme. For these reasons, we favor the model proposed here over the prior model.
The results of this study also provided the surprising finding that when introduced into yeast mitochondria, whereas the chloroplast regulatory region impairs oxidative phosphorylation, it impairs cytochrome c oxidase but not the ATP synthase or Complex I or III of the electron transport chain (Tables 1 and  2). This suggests that the ATP synthase either directly or indirectly interacts with cytochrome c oxidase. The interaction of the ATP synthase appears to be guided by interaction directly with the cysteine residues in the regulatory region, because serines at these positions have no effect. Alternatively, the oxidation of these sites might cause a conformational change that is required for the yet unidentified interaction. Although the conformational specific interaction seems unlikely, it is supported by the fact that the smaller chimeric constructs did not have that same effect on cytochrome c oxidase despite the presence of the cysteine residues.
There have been other reports on the possible interaction of the ATP synthase with cytochrome c oxidase. Deletion of subunit g of the yeast ATP synthase has been shown to both decrease the formation of the dimer form of the ATP synthase and reduce the level of cytochrome c oxidase (20,41,42). In addition, electron paramagnetic resonance and differential scanning calorimetric studies have indicated a direct interaction between cytochrome c oxidase and the ATP synthase (43). Thus, in conjunction with the results of this study, there is growing evidence that the ATP synthase interacts with cytochrome c oxidase, possibly forming a supercomplex. However, the results in this study still do not exclude the possibility that the reduction in cytochrome c oxidase is due to an interaction with a component involved in the assembly or synthesis of cytochrome c oxidase. Current studies are underway to clarify this further.