Definition of the Interaction Domain for Cytochrome c on the Cytochrome bc 1 Complex STEADY-STATE AND RAPID KINETIC ANALYSIS OF ELECTRON TRANSFER BETWEEN CYTOCHROME c AND RHODOBACTER SPHAEROIDES CYTOCHROME bc 1 SURFACE MUTANTS

The interaction domain for cytochrome c on the cytochrome bc 1 complex was studied using a series of Rhodobacter sphaeroides cytochrome bc 1 mutants in which acidic residues on the surface of cytochrome c 1 were substituted with neutral or basic residues. Intra-complex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyri-dine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc 1 at low ionic strength resulted in electron transfer from pho-toreduced heme c to cytochrome c 1 with a rate constant of k et 5 6 3 10 4 s 2 1 . Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k et values as well as significantly higher equilibrium dissociation constants and steady-state K m values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c 1 are involved in binding positively charged cytochrome c . These acidic residues on the intramembrane surface of cytochrome c 1 direct the diffusion and binding of

The cytochrome bc 1 complex is an essential component of the energy-transducing electron transfer chains in mitochondria and many prokaryotes (1). The complex from all organisms contains two b cytochromes (b L and b H ) located on a single polypeptide chain, cytochrome c 1 , and the Rieske iron-sulfur protein. The total number of polypeptide subunits depends on the species, ranging from three in some prokaryotes to 11 in beef heart mitochondrial cytochrome bc 1 . It is generally accepted that electron transfer through the complex involves a Q-cycle mechanism in which four protons are translocated from the negative to the positive side of the membrane per two electrons transferred from ubiquinol to cytochrome c (2). The electron transfer reaction between cytochrome c and the cytochrome bc 1 complex involves, at a minimum, the following steps: 1) formation of a 1:1 reactant complex between cytochrome c 3ϩ and reduced cytochrome bc 1 , 2) intracomplex elec-tron transfer from cytochrome c 1 2ϩ to cytochrome c 3ϩ , and 3) dissociation of the product complex to release cytochrome c 2ϩ . The binding interaction between cytochromes c and bc 1 must stabilize the optimal orientation of the reactant complex for rapid electron transfer and allow both rapid reactant complex formation and product complex dissociation. Both steady-state and stopped-flow kinetic studies have shown that the reaction rate is inhibited by high ionic strength, indicating that the interaction has an electrostatic component (3)(4)(5). Extensive chemical modification studies have revealed that six or seven lysine amino groups surrounding the heme crevice of cytochrome c are involved in binding cytochrome bc 1 (6 -9). Studies utilizing a water-soluble carbodiimide have implicated the acidic residues 66, 67, 76, and 77 on bovine cytochrome c 1 in cytochrome c binding as well as acidic residues on the hinge protein (10,11). Acidic residues in sequence 165-174 have also been implicated in cytochrome c binding by photoaffinity cross-linking studies (12). The x-ray crystal structures of beef and chicken cytochromes bc 1 have revealed that the exposed heme CD edge of cytochrome c 1 on the cytoplasmic surface of the membrane is surrounded by acidic residues that could form a docking site for cytochrome c (13)(14)(15). The hinge protein (subunit 8) is located adjacent to cytochrome c 1 , and acidic residues on its cytoplasmic surface could help direct cytochrome c to the docking site.
In this paper, the interaction domain for cytochrome c on the cytochrome bc 1 complex was defined using a series of Rhodobacter sphaeroides cytochrome bc 1 mutants in which acidic residues on the surface of cytochrome c 1 were substituted with neutral or basic residues. Steady-state kinetic studies were carried out to determine how mutation affected V max and K m for cytochrome c. A new ruthenium photoexcitation technique was also used to resolve a key step in the mechanism, intracomplex electron transfer between cytochrome c and purified cytochrome c 1 (16,17). In this technique, a 1:1 electrostatic complex between cytochrome bc 1 and a cytochrome c derivative labeled with ruthenium trisbipyridine is formed at low ionic strength. The Ru(II) group is photoexcited to a metalto-ligand charge transfer state, Ru(II)*, which is a strong reducing agent and rapidly transfers an electron to heme c (18). Electron transfer between photoreduced heme c and cytochrome c 1 can be measured on a microsecond time scale (17). Electron transfer between ruthenium trisbipyridine-labeled cytochrome c and the cytochrome bc 1 complex isolated from bovine mitochondria as well as wild-type and mutant R. sphaeroides was studied over a wide range of conditions.

EXPERIMENTAL PROCEDURES
Materials-Bovine cytochrome bc 1 was purified as described by Yu et al. (19). Horse heart cytochrome c was obtained from Sigma. Horse heart Ru-72-Cc, 1 labeled with ruthenium trisbipyridine at lysine 72, was prepared as described by Durham et al. (18). Cytochrome oxidase was purified as described by Capaldi and Hayashi (20). Aniline was purchased from Fisher, and 3CP was purchased from Aldrich. Stock solutions of aniline were prepared fresh daily. A stock solution of 3CP was adjusted to pH 7 using NaOH and was stored at Ϫ70°C. Following mutagenesis, a 1.2-kilobase XbaI-HindIII fragment from pSELfbcFBC H Q (21) containing the altered codon was ligated into the XbaI and HindIII sites of pRKDfbcFBC km Q. The use of pRKDfbcFB-C km Q to receive the mutated XbaI-HindIII fragment eliminates the possibility of retaining or recloning the wild-type fragment when attempting to subclone the mutated fragments into the expression vector. Loss of kanamycin resistance was then used to screen for recombinant plasmids. pRKDfbcFBC H Q derivatives were then conjugated into R. sphaeroides BC17 from Escherichia coli S17-1 using a plate-mating procedure. The His-tagged cytochrome bc 1 mutant complexes were prepared essentially according to the method described by Tian et al. (21).

Construction of Mutations and Expression of Mutant Complexes in R. sphaeroides-Mutants
Steady-state Assay for the Reaction between Cytochromes c and bc 1 -The purified cytochrome bc 1 complex was diluted to a final cytochrome b concentration of 3 M with buffer containing 50 mM Tris-Cl (pH 8), 100 mM NaCl, and 0.01% dodecyl maltoside. 3 l of diluted bc 1 complex was added to a 1-ml assay mixture containing 25 mM sodium/potassium phosphate buffer (pH 7.4), 0.3 mM EDTA, 150 mM NaCl, 25 M Q 0 C 10 Br, and various amounts of cytochrome c (2-120 M). Steady-state activity was measured from the rate of reduction of cytochrome c detected at 550 nm using a millimolar extinction coefficient of 18.5 cm Ϫ1 . The rate of nonenzymatic reduction of cytochrome c by Q 0 C 10 BrH 2 , determined under the same conditions in the absence of enzyme, was subtracted. The steady-state parameters V max and K m were determined from Eadie-Hofstee plots of the initial velocities as described (22).
Flash Photolysis Experiments-Transient absorbance measurements were carried out as described by Heacock et al. (17) by flash photolysis of 300-l solutions contained in a 1-cm glass semimicrocuvette. The excitation light pulse was provided by a phase R Model DL1400 flash lamp-pumped dye laser using coumarin LD 490 to produce a 480-nm light flash of Ͻ0.5-s duration. The detection system has been described by Heacock et al. (17). The reactions of cytochromes c and c 1 were monitored over the wavelength range of 540 -560 nm using samples containing 5-20 M cytochrome bc 1 , 5-10 M Ru-72-Cc, 50 nM cytochrome c oxidase (catalytic amount), 10 mM aniline, 1 mM 3CP, and 0.01% lauryl maltoside in 5 mM Tris-Cl (pH 7.4). Aniline and 3CP were used as sacrificial electron donors to reduce Ru(III) and to prevent the back-reaction between Ru(III) and Fe(II) of cytochrome c. All absorbance transients were analyzed using the KINFIT kinetics program obtained from On-Line Instrument Systems, Inc. The absorbance spectra were obtained with a Hewlett-Packard 8452A diode array spectrophotometer.

Intracomplex Electron Transfer between Ru-72-Cc and Bo-
vine Cytochrome bc 1 -The electron transfer reaction between cytochrome c and bovine cytochrome bc 1 was studied using the ruthenium photoreduction technique previously described (17). Horse Ru-72-Cc was chosen because it has a high efficiency of photoreduction in a single flash (35%) and because it interacts favorably with purified bovine cytochrome c 1 (17). Flash photolysis of a solution containing 9 M Ru-72-Cc and 10 M bovine cytochrome bc 1 in low ionic strength buffer resulted in rapid electron transfer from Ru(II)* to cytochrome c Fe(III) as indicated by the rapid increase in absorbance at 548 nm ( Fig. 1). Aniline and 3CP were present in the solution to reduce Ru(III) and to prevent the back-reaction k 2 (Scheme 1). The rapid increase in absorbance at 548 nm was followed by an exponential decrease with a rate constant of 6.0 ϫ 10 4 s Ϫ1 , indicating electron transfer from cytochrome c Fe(II) to cytochrome c 1 Fe(III). The reduction of cytochrome c 1 was observed directly at 556.5 nm, which is an isosbestic point of cytochrome c. The exponential increase in absorbance at 556 nm has the same rate constant as the 548 nm transient, 6.0 ϫ 10 4 s Ϫ1 . Transients recorded over the wavelength range of 540 -560 nm yielded a kinetic difference spectrum with a maximum at 546 nm and a minimum at 556 nm (Fig. 2), consistent with the difference spectrum for electron transfer between cytochromes c and c 1 (3).

SCHEME 1
Electron Transfer between Cytochromes c and bc 1 was taking place in a bound complex between Ru-72-Cc and cytochrome bc 1 with a dissociation constant of Ͻ4 M, according to the top line of Scheme 2. The rate constant remained the same (6.0 ϫ 10 4 s Ϫ1 ) as the ionic strength was increased from 5 to 45 mM (Fig. 3). A second slow phase first appeared at 35 mM ionic strength with a relative amplitude of 36% and increased to 60% relative amplitude at 45 mM ionic strength with a rate constant of 6100 s Ϫ1 (Fig. 3). The fast phase was completely eliminated above 55 mM ionic strength, indicating dissociation of the Ru-72-Cc⅐cytochrome bc 1 complex. The slow phase obeyed second-order kinetics, indicating that it was due to a reaction between solution Ru-72-Cc and cytochrome bc 1 according to Scheme 2. The rate constant of the slow phase decreased with further increases in ionic strength above 55 mM, consistent with a reaction between oppositely charged proteins.
Electron Transfer between Ru-72-Cc and Wild-type R. sphaeroides Cytochrome bc 1 -Flash photolysis of a solution containing 9 M Ru-72-Cc and 10 M R. sphaeroides cytochrome bc 1 at low ionic strength led to a rapid increase in 548 nm absorbance, followed by an exponential decrease with a rate constant of 6.0 ϫ 10 4 s Ϫ1 due to electron transfer from the cytochrome c heme Fe(II) to cytochrome c 1 . The 556 nm transient also had a rate constant of 6 ϫ 10 4 s Ϫ1 , consistent with reduction of cytochrome c 1 . The rate constant was independent of protein concentration, indicating that the reaction was due to intracomplex electron transfer. The kinetic difference spectrum was nearly the same as for the complex with beef cytochrome bc 1 (Fig. 2). The rate constant remained nearly the same as the ionic strength was increased to 45 mM; then the amplitude of the fast intracomplex phase decreased, and a new slow phase appeared (Fig. 4). The fast and slow phases were of equal amplitude at 65 mM ionic strength, with a rate constant of 5200 s Ϫ1 for the slow phase. The fast phase was completely eliminated above 80 mM ionic strength, indicating dissociation of the complex. The rate constant of the slow phase was linearly dependent on the concentration of cytochrome bc 1 (data not shown), indicating bimolecular kinetics according to Scheme 2 with a second-order rate constant of k 2 ϭ 3.1 ϫ 10 8 M Ϫ1 s Ϫ1 at 85 mM ionic strength. The rate constant of the slow phase decreased with increasing ionic strength above 65 mM (Fig. 4).
To Characterization of R. sphaeroides Cytochrome bc 1 Surface Mutants by Steady-state Kinetics-Acidic residues distributed along the surface of the cytochrome c 1 subunit were mutated to neutral or basic residues to define the docking interface for cytochrome c. All of the mutants grew photosynthetically at rates comparable to that of the complement strain. The steadystate reactions of horse cytochrome c with the cytochrome bc 1 mutants were carried out at high ionic strength as described above. Wild-type cytochrome bc 1 and each of the mutants displayed monophasic kinetics with well defined V max and K m values ( Fig. 5 and Table I). The largest effect on the steadystate kinetics was observed for the double mutant E100K/ E101K, which has a K m value of 73 M, ϳ7-fold larger than that of wild-type cytochrome bc 1 (Fig. 5 and Table I). Mutants E74Q, E104Q, D109N, and E162Q/E163Q each have a K m value that is ϳ5-fold larger than that of wild-type cytochrome bc 1 ( Table  I). The E169Q mutation led to a 3-fold increase in K m . The increase in K m for these mutants indicates that the binding affinity for cytochrome c has decreased. However, the steadystate V max values for these mutants are comparable to that for the wild-type enzyme. This observation partially explains why the growth rate is not impaired by these mutations since the steady-state enzymatic activity at high cytochrome c concentration is comparable to that of wild-type cytochrome bc 1 . Mu-tants E56Q, E79Q, E82Q/D83N, D95K, E97K/E98K, D213K/ D214N, D214K, E217K, D220K, and D223N have K m and V max values comparable to those of the wild-type strain (Table I).
Reaction between Ru-72-Cc and R. sphaeroides Cytochrome bc 1 Surface Mutants-The binding interaction was also defined by studying the reactions of Ru-72-Cc with the R. sphaeroides cytochrome bc 1 surface mutants. All of the mutants displayed intracomplex electron transfer with Ru-72-Cc at 5 mM ionic strength. The intracomplex rate constants for electron transfer    Electron Transfer between Cytochromes c and bc 1 from heme c to cytochrome c 1 ranged from 1.2 ϫ 10 4 s Ϫ1 for E162K/E163Q to 7.0 ϫ 10 4 s Ϫ1 for E56K (Table I) Table I). The fast intracomplex phase for electron transfer was eliminated at a much lower ionic strength for these mutants than for wild-type cytochrome bc 1 (Figs. 4 and 6 -8 and Table I). For example, the fast intracomplex phase of electron transfer was completely eliminated at 25 mM ionic strength for the E101K/D102K mutant and at 45 mM ionic strength for the E104Q mutant ( Figs. 4 and 8). The percent of the fast intracomplex phase of electron transfer at 45 mM ionic strength was 30% or less for the E74Q, D95K, E100K/ D101K, E104Q, D109N, E162Q/E163Q, and E169Q, mutants, compared with 80% for wild-type cytochrome bc 1 (Fig. 8 and Table I). The rate constant of the slow phase due to the bimolecular reaction was less than that of wild-type cytochrome bc 1 for these mutants and decreased with increasing ionic strength (Figs. 4, 6, and 7). Mutations at Glu-56, Glu-79, Glu-82, Asp-83, Glu-97, Glu-98, Asp-213, Asp-214, Asp-220, and Asp-223 had relatively minor effects on the rate constant for intracomplex electron transfer and the percent of the fast phase at 45 mM ionic strength, indicating that these residues are not involved in binding cytochrome c (Table I).

Intracomplex Electron Transfer between Ru-72-Cc and Cytochrome bc 1 -
The reaction between cytochrome c and the cytochrome bc 1 complex involves the following series of steps: 1) diffusion of ferricytochrome c to cytochrome bc 1 to form a transient reactant complex, 2) electron transfer within the reactant complex to form a product complex, and 3) dissociation of the product complex to release ferrocytochrome c. Extensive steady-state and stopped-flow spectroscopic studies have revealed many important features of this reaction (3-7, 9). However, it has not been possible to measure the rate constants of individual steps in the overall mechanism using these tech-niques. The ruthenium photoexcitation technique has made it possible to measure the rate constant for a key step in this mechanism, intracomplex electron transfer between cytochrome c and purified cytochrome c 1 (17). In this investigation, the reactions between cytochrome c and bovine and R. sphaeroides cytochromes bc 1 were studied using horse Ru-72-Cc. This derivative is the best currently available for this application. The rate constant for electron transfer from Ru(II)* to heme c in Ru-72-Cc is k 1 ϭ 3 ϫ 10 7 s Ϫ1 , which is very fast compared with intracomplex electron transfer between heme c and cytochrome c 1 (18). The yield of heme c reduced with a single laser flash is 35%, which is among the largest for the available ruthenium trisbipyridine-labeled cytochrome c deriv- atives. This ensures that electron transfer between cytochromes c and c 1 can be detected with good signal-to-noise ratio. This reaction is difficult to detect because the spectra of cytochromes c and c 1 are very similar, leading to small absorbance changes. Ru-72-Cc has a 3-fold smaller V max and a 1.5-fold higher K m than native horse cytochrome c in the steady-state reaction with R. sphaeroides cytochrome bc 1 at high ionic strength. This indicates that the ruthenium complex does have an effect on the interaction with cytochrome c 1 , consistent with the location of Lys-72 to the left side of the heme crevice. However, this effect is not too large, and Ru-72-Cc forms a strong 1:1 complex with cytochrome bc 1 at low ionic strength. Previous studies have shown that among the ruthenium trisbipyridine-labeled cytochrome c derivatives that could be used for this application, Ru-72-Cc has the largest intracomplex electron transfer rate constant and binding constant for the reaction with purified beef cytochrome c 1 (17). Unfortunately, the photoreduction yield is too small to detect intracomplex electron transfer for those ruthenium trisbipyridine-labeled cytochrome c derivatives that are labeled on the back of cytochrome c and have the same binding constant as native cytochrome c.
Flash photolysis of a 1:1 complex between Ru-72-Cc and bovine cytochrome bc 1 at low ionic strength results in electron transfer from photoreduced heme c to cytochrome c 1 with a rate constant of 6.0 ϫ 10 4 s Ϫ1 . The kinetic difference spectrum has the same wavelength dependence as the difference spectrum for electron transfer between native cytochrome c 1 and cytochrome c obtained by equilibrium mixing techniques (3). The kinetic difference spectrum reflects the extent of the reaction, controlled by the equilibrium constant K eq , as well as the spectra of the two components. The equilibrium constant for the electron transfer reaction between purified ferrocytochrome c 1 and ferricytochrome c is K eq ϭ 3.3 at high ionic strength (3), consistent with redox potentials of 228 and 261 mV for the two cytochromes, respectively. The reduction potential of cytochrome c decreases to 220 mV upon complex formation with cytochrome oxidase (23). If a similar change occurs upon complex formation with cytochrome bc 1 , this would lead to a decrease in K eq and a larger kinetic difference spectrum for the reverse electron transfer reaction measured in the present experiments. Complex formation may also lead to slight changes in the ␣-bands of the two cytochromes (17,24). Since the spectra of cytochromes c and c 1 are so similar to each other, very slight spectral changes could lead to larger changes in the kinetic difference spectrum.
Ionic Strength Dependence of the Reaction between Ru-72-Cc and Cytochrome bc 1 -The reactions of cytochrome c with its redox partners in mammalian mitochondria are thought to occur by a three-dimensional diffusion process in the intermembrane space under physiological conditions of 100 -150 mM ionic strength (25). We therefore studied the reaction of Ru-72-Cc with cytochrome bc 1 as the conditions were changed continuously from low ionic strength, where intracomplex electron transfer was observed, to high ionic strength, where bimolecular kinetics occurred. The rate constant for intracomplex electron transfer between Ru-72-Cc and bovine cytochrome bc 1 remains constant at k et ϭ 6.0 ϫ 10 4 s Ϫ1 as the ionic strength is increased from 5 to 45 mM. This indicates that the orientation of the 1:1 complex is optimal for electron transfer at low ionic strength and does not change as the ionic strength is increased. The rate constant for intracomplex electron transfer with R. sphaeroides cytochrome bc 1 is also nearly independent of ionic strength. Similar behavior has also observed in the reactions of cytochrome c with purified cytochrome c 1 (17), cytochrome oxidase (16,27), cytochrome c peroxidase (28 -31), and cytochrome b 5 (32,33).
As the ionic strength is increased above 35 mM, the amplitude of the fast intracomplex phase decreases, and a new slow phase appears due to bimolecular electron transfer between solution Ru-72-Cc and cytochrome bc 1 . The relative amplitudes of the fast and slow phases can be used to estimate the equilibrium dissociation constant K D ϭ k d /k f for the 1:1 complex to be 9.5 M at 45 mM ionic strength for bovine cytochrome bc 1 and 5.5 M at 65 mM ionic strength for R. sphaeroides cytochrome bc 1 . The presence of both the fast intracomplex phase and the slow bimolecular phase at intermediate ionic strength provides strong evidence that the complete bimolecular reaction involves formation of a 1:1 complex, followed by intracomplex electron transfer according to Scheme 2. The dissociation rate constant k d must be much smaller than k et since if k d were larger than k et , then rapid equilibrium conditions would apply, and separate slow and fast phases would not be observed (31). Assuming that the bimolecular reaction obeys Scheme 2 and k d Ͻ Ͻ k et , the observed rate constant is given by Equation 1 (31), where E 0 is the concentration of cytochrome bc 1 and C 0 is the concentration of Ru-72-Cc. From this equation, the formation and dissociation rate constants can be estimated to be k f ϭ 9.6 ϫ 10 8 M Ϫ1 s Ϫ1 and k d ϭ 9.1 ϫ 10 3 s Ϫ1 at 45 mM ionic strength for bovine cytochrome bc 1 and k f ϭ 9.5 ϫ 10 8 M Ϫ1 s Ϫ1 and k d ϭ 5.2 ϫ 10 3 s Ϫ1 at 65 mM ionic strength for R. sphaeroides cytochrome bc 1 . The complex formation rate constant of 10 9 M Ϫ1 s Ϫ1 is consistent with an electrostatically assisted, diffusion-controlled reaction between cytochrome c and the negatively charged surface of cytochrome c 1 . Similar complex formation rate constants have been measured for the reactions of cytochrome c with cytochrome c peroxidase (31), cytochrome c oxidase (27), and cytochrome b 5 (32,33). The overall reaction between Ru-72-Cc and bovine cytochrome bc 1 at 45 mM ionic strength thus involves formation of a 1:1 cytochrome c⅐bc 1 complex with rate constant k f ϭ 9.6 ϫ 10 8 M Ϫ1 s Ϫ1 , intracomplex electron transfer with rate constant k et ϭ 6.0 ϫ 10 4 s Ϫ1 , and product complex dissociation with rate constant k d ϭ 9.1 ϫ 10 3 s Ϫ1 .
The second-order rate constant for the reaction between Ru-72-Cc and both bovine and R. sphaeroides cytochromes bc 1 decreases rapidly with increasing ionic strength. At ionic strengths above 100 mM, it is likely that k d will become comparable to k et and that k 2 will be a function of all three rate constants in Scheme 2, as given in Equation 2.
The decrease in k 2 with increasing ionic strength is most likely due to a decrease in k f as well as an increase in k d . k et is unlikely to change very much at high ionic strength since it is unchanged from 5 to 65 mM ionic strength. The large ionic strength dependence of k 2 is an indication of the strong electrostatic interaction between cytochromes c and bc 1 .

Definition of the Cytochrome c-binding Domain by Kinetic Studies of R. sphaeroides Cytochrome bc 1 Surface Mutants-
Among the three redox-active subunits of cytochrome bc 1 from different species, the cytochrome c 1 subunit is the least conserved. Except for the heme ligands (His-41 and Met-160 in the bovine sequence), heme anchors (Cys-37 and Cys-40), Arg-120 (which forms a salt bridge with heme c propionate), and the heme-embracing sequence -PDL-(-ADL-in R. sphaeroides), there is no conserved sequence with known function. Both bovine and R. sphaeroides cytochrome c 1 subunits belong to Ambler's class I cytochrome c family. Three conserved helices, namely ␣1, ␣2, and ␣5 in horse cytochrome c and other types of class I cytochromes, are also present in chicken and bovine cytochromes c 1 with the same orientation relative to each other and essentially occupy the same space. These three helices form the functional domains of cytochrome c 1 . Fig. 9 shows the sequence alignment of bovine and R. sphaeroides cytochrome c 1 subunits, and essentially aligns the conserved secondary structure in all type I cytochromes c and the conserved residues mentioned above. The major differences between R. sphaeroides and bovine cytochromes c 1 are the deletion and insertions in the loop regions. R. sphaeroides cytochrome c 1 has a 10-amino acid deletion around the ␣1Љ helix of bovine sequence 100 -110, a 16-amino acid insertion (residues 132-147 in R. sphaeroides) before the conserved ␣3 helix, and a 21-residue insertion (residues 181-201) before conserved heme ligand Met-207. The x-ray crystal structure of bovine cytochrome bc 1 shows that the surface acidic residues on cytochrome c 1 are clustered in three regions (Figs. 9 and 10). Region 1 consists of ␣1Ј (residues 52-69 in the bovine sequence) and the following loop (residues 70 -90 in the bovine sequence), which is involved in dimer contact with cytochrome c 1 from the other monomer. Region 2 is loop Met-160 to ␣5 (residues 160 -178), and region 3 is loop ␣3 to ␤1 (residues 132-146). Region 1 has been identified as part of the cytochrome c-binding domain by carbodiimide modification studies (10,11), whereas region 2 has been implicated in cytochrome c binding by photoaffinity cross-linking studies (12). The amino terminus of subunit 8, consisting of eight consecutive glutamate residues, was also found to be important in binding cytochrome c in the carbodiimide modification studies (10,11). However, no electron density was observed for the N-terminal 14 residues of subunit 8, indicating that this segment is highly mobile.
The 16-residue insertion (residues 132-147) before the ␣3 helix in R. sphaeroides cytochrome c 1 does not contain any charged residues, indicating that this part is most likely buried in the protein. This is consistent with the relative location of the N terminus of the ␣3 helix, which is buried deep in the protein (Fig. 10). Compared with bovine cytochrome c 1 , the loop connecting the ␣3 helix and heme ligand Met-207 in R. sphaeroides cytochrome c 1 contains five more acidic residues (total is seven) and is 21 amino acids longer in length. Only two acidic residues are located in this loop in bovine cytochrome bc 1 (Fig.  10), which interacts with residues 50 -60 of subunit 8 (containing six acidic residues). Since there is no subunit 8 in R. sphaeroides, the five extra acidic residues in the loop connecting ␣3 and heme ligand Met-207 may perform the function of subunit 8. Chemical modification and ionic strength dependence studies have shown that the electrostatic interaction of both horse cytochrome c and R. sphaeroides cytochrome c 2 with R. sphaeroides cytochrome bc 1 is very similar to that of horse cytochrome c with bovine cytochrome bc 1 (6 -8, 22, 34).
Acidic residues in each of the three acidic regions on the surface of R. sphaeroides cytochrome c 1 were mutated to neutral or basic residues to define the interaction domain for cytochrome c. Mutations near bovine residue 79 in region 1 and residue 136 in region 3 (residues 100 -109 and 162-169 in the R. sphaeroides sequence) had significant effects on the intracomplex electron transfer rate k et as well as on the steady-state Michaelis constant K m for cytochrome c (Fig. 10B and Table I). Mutations E74Q, E101K/D102K, E104Q, and D109N in region 1 and E162Q/E163Q and E169Q in region 3 each decreased k et by 2-5-fold compared with wild-type cytochrome bc 1 (Table I). This decrease in k et indicates that Ru-72-Cc binds to the mutant cytochrome bc 1 in an orientation that is not as favorable for electron transfer as that of wild-type cytochrome bc 1 at low ionic strength. These mutations also led to a decrease in the ionic strength for the transition between intracomplex electron transfer and bimolecular electron transfer (Figs. 4 and 6 -8). The percent of the fast intracomplex phase of electron transfer was 30% or less for the above mutants at 45 mM ionic strength, compared with 80% for wild-type cytochrome bc 1 (Fig. 8 and Table I). These mutations therefore increase the equilibrium dissociation constant K D , consistent with the increase in steady-state K m . The mutations listed above also led to a decrease in the second-order rate constant at high ionic strength, paralleling the effects on k et , K D , and K m (Figs. 4, 6, and 7). Not all of the acidic residues in region 1 are involved in binding cytochrome c, however, since mutations E79Q, E82Q/D83N, and E97K/E98K in this region had no effect on the steady-state or rapid kinetic parameters. The D95K mutation had a significant effect on the rapid kinetic parameters, but only a small effect on the steady-state K m , suggesting a marginal role in binding. None of the mutations in acidic region 2 (residues 212-223 in the R. sphaeroides sequence) had a significant effect on the steady-state or rapid kinetics (Table I), indicating that acidic residues in region 2 are not involved in binding cytochrome c. However, Broger et al. (12) reported that the photoaffinity analogue arylazidolysine 13-cytochrome c was crosslinked to bovine cytochrome c 1 somewhere in region 2, suggesting that this region may by close to the cytochrome c-binding site. The ruthenium complex may cause Ru-72-Cc to bind to a somewhat different domain on the surface of cytochrome c 1 than native horse cytochrome c. However, the charge mutations have nearly the same pattern of effects on the rapid kinetics with Ru-72-Cc as on the steady-state kinetics with native horse cytochrome c, indicating that any change in the binding domain is small.
The mutational studies indicate that acidic residues in regions 1 and 3 of R. sphaeroides cytochrome c 1 are involved in binding positively charged cytochrome c. Regions 1 and 3 are located on the intramembrane surface of bovine cytochrome c 1 near the exposed CD edge of the heme. These two regions on opposite sides of the heme crevice appear to direct the diffusion and binding of cytochrome c from the intramembrane space. The region immediately surrounding the heme crevice is relatively hydrophobic. It thus appears that the interaction between cytochromes c and bc 1 consists of a central hydrophobic domain surrounded by complementary electrostatic interactions at the periphery. A similar type of interaction domain is observed in the complex between yeast cytochrome c and cytochrome c peroxidase, which has been characterized by x-ray crystallography and mutagenesis studies (26,30,31).  10. A, structure of cytochrome c 1 and subunit 8 of bovine heart mitochondria, viewed parallel to the membrane. Acidic residues exposed to the intramembrane space region are shown in red, and the three acidic regions identified in the legend to Fig. 9 are boxed. The heme, heme ligands (His-41 and Met-160), and the conserved heme-embracing tripeptide -PDL-are displayed in a stick model. The heme A-D edge, where electron transfer between the iron-sulfur protein and cytochrome c 1 is thought to occur, is identified. B, structure of cytochrome c 1 and subunit 8 viewed from the intramembrane space down the membrane. Acidic residues exposed to the surface of cytochrome c 1 are colored red and labeled. The conserved R. sphaeroides acidic residues are indicated in parentheses. The heme group is shown as a space-filling model colored red. The coordinates for bovine cytochrome c 1 were provided by Dr. Di Xia (personal communication).