A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.     

Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway

Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O(2) to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O(2) reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O(2) reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O(2) reduction, but a decrease in the apparent pK(a) of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.     

Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pK(a) values associated with the pH dependencies of specific proton transfer (PT) reactions in CcO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent pK(a) of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated pK(a) values of Glu286 at these different conformations to reproduce the experimentally observed apparent pK(a) of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans CcO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in CcO.     

Mutations which decouple the proton pump of the cytochrome c oxidase from Rhodobacter sphaeroides perturb the environment of glutamate 286

Mutants that decouple the proton pump of cytochrome c oxidase from Rhodobacter sphaeroides are postulated to do so by increasing the pK(a) of glutamate 286, which is 20 Angstrom away. The possibility that a conformational change near E286 is induced by the decoupling mutations (N139D and N207D) was investigated by FTIR difference spectroscopy. In both decoupled mutants, the reduced-minus-oxidized FTIR difference spectra show a shift of 2 cm(-1) to lower frequency of the band resulting from the absorbance of E286 in the oxidized enzyme. The decoupling mutants may influence E286 by altering the chain of water molecules which runs from the site of the mutations to E286.     

Tracing the D-pathway in reconstituted site-directed mutants of cytochrome c oxidase from Paracoccus denitrificans

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.     

Time-resolved ATR-FTIR spectroscopy of the oxygen reaction in the D124N mutant of cytochrome c oxidase from Paracoccus denitrificans

Real-time measurements of the cytochrome c oxidase reaction with oxygen were performed by ATR-FTIR spectroscopy, using a mutant with a blocked D-pathway of proton transfer (D124N, Paracoccus denitrificans numbering). The complex spectrum of the ferryl-->oxidized transition together with other bands showed protonation of Glu 278 with a peak position at 1743 cm-1. Since our time resolution was not sufficient to follow the earlier reaction steps, the FTIR spectrum of the CO-inhibited fully reduced-->ferryl transition was obtained as a difference between the spectrum before the laser flash and the first spectrum after it. A trough at 1735 cm-1 due to deprotonation of Glu 278 was detected in this spectrum. These observations confirm the proposal [Smirnova I.A., et al. (1999) Biochemistry 38, 6826-6833] that the proton required for chemistry at the binuclear site is taken from Glu 278 in the perroxy-->ferryl step, and that the rate of the next step (ferryl-->oxidized) is limited by reprotonation of Glu 278 from the N-side of the membrane in the D124N mutant enzyme. The blockage of the D-pathway in this mutant for the first time allowed direct detection of deprotonation of Glu 278 and its reprotonation during oxidation of cytochrome oxidase by O2.     

Electrostatic environment of hemes in proteins: pK(a)s of hydroxyl ligands

The pK(a)s of ferric aquo-heme and aquo-heme electrochemical midpoints (E(m)s) at pH 7 in sperm whale myoglobin, Aplysia myoblogin, hemoglobin I, heme oxygenase 1, horseradish peroxidase and cytochrome c oxidase were calculated with Multi-Conformation Continuum Electrostatics (MCCE). The pK(a)s span 3.3 pH units from 7.6 in heme oxygenase 1 to 10.9 in peroxidase, and the E(m)s range from -250 mV in peroxidase to 125 mV in Aplysia myoglobin. Proteins with higher in situ ferric aquo-heme pK(a)s tend to have lower E(m)s. Both changes arise from the protein stabilizing a positively charged heme. However, compared with values in solution, the protein shifts the aquo-heme E(m)s more than the pK(a)s. Thus, the protein has a larger effective dielectric constant for the protonation reaction, showing that electron and proton transfers are coupled to different conformational changes that are captured in the MCCE analysis. The calculations reveal a breakdown in the classical continuum electrostatic analysis of pairwise interactions. Comparisons with DFT calculations show that Coulomb's law overestimates the large unfavorable interactions between the ferric water-heme and positively charged groups facing the heme plane by as much as 60%. If interactions with Cu(B) in cytochrome c oxidase and Arg 38 in horseradish peroxidase are not corrected, the pK(a) calculations are in error by as much as 6 pH units. With DFT corrected interactions calculated pK(a)s and E(m)s differ from measured values by less than 1 pH unit or 35 mV, respectively. The in situ aquo-heme pK(a) is important for the function of cytochrome c oxidase since it helps to control the stoichiometry of proton uptake coupled to electron transfer [Song, Michonova-Alexova, and Gunner (2006) Biochemistry 45, 7959-7975].     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

Proton titration curve of yeast iso-1-ferricytochrome c. Electrostatic and conformational effects of point mutations.

The proton titration curves of yeast iso-1-ferricytochrome c and selected point mutants of this protein have been determined between pH 3 and 11 at 10 and 25 degrees C with a computer-controlled titration system. Initial titration of the wild-type protein to acidic pH followed by subsequent titrations to alkaline and then acidic pH demonstrates hysteresis, with one more group (28.7) titrating between pH 11 and 3 than originally titrated (27.7) between pH 3 and 11. Initial titration to alkaline pH, however, resulted in observation of the same number of groups in both directions of titration (28.7 vs 28.6). At 10 degrees C, 7.5 fewer groups were found to titrate over the same range of pH. Titration curves obtained for six cytochrome c mutants modified at Arg-38, Phe-82, Tyr-48, and Tyr-67 were analyzed by subtraction of the corresponding titration curve for the wild-type protein to produce difference titration curves. In most cases, the effects of these mutations as revealed in the difference titration curves could be accounted for as either the result of introduction of an additional group titrating within this pH range, the result of a change in the pK of a titrating residue, and/or the result of a change in the pK for either the first acidic or the first alkaline protein conformational transition. In addition to demonstration of the electrostatic consequences of the mutations in cytochrome c studied here, this study establishes the general usefulness of precise proton titration curve analysis in the characterization of variant proteins produced through recombinant genetic techniques.     

Subunit III of cytochrome c oxidase of Rhodobacter sphaeroides is required to maintain rapid proton uptake through the D pathway at physiologic pH

The catalytic core of cytochrome c oxidase is composed of three subunits where subunits I and II contain all of the redox-active metal centers and subunit III is a seven transmembrane helix protein that binds to subunit I. The N-terminal region of subunit III is adjacent to D132 of subunit I, the initial proton acceptor of the D pathway that transfers protons from the protein surface to the buried active site approximately 30 A distant. The absence of subunit III only slightly alters the initial steady-state activity of the oxidase at pH 6.5, but activity declines sharply with increasing pH, yielding an apparent pK(a) of 7.2 for steady-state O(2) reduction. When subunit III is present, cytochrome oxidase is more active at higher pH, and the apparent pK(a) of steady-state O(2) reduction is 8.5. Single-turnover experiments show that proton uptake through the D pathway at pH 8 slows from >10000 s(-1) in the presence of subunit III to 350 s(-1) in its absence. At low pH (5.5) the D pathway of the oxidase lacking subunit III regains its capacity for rapid proton uptake. Analysis of the F --> O transition indicates that the apparent pK(a) of the D pathway in the absence of subunit III is 6.8, similar to that of steady-state O(2) reduction (7.2). The pK(a) of D132 itself may decline in the absence of subunit III since its carboxylate group will be more exposed to solvent water. Alternatively, part of a proton antenna for the D pathway may be lost upon removal of subunit III. It is proposed that one role of subunit III in the normal oxidase is to maintain rapid proton uptake through the D pathway at physiologic pH.     

Role of Asp1393 in catalysis, flavin reduction, NADP(H) binding, FAD thermodynamics, and regulation of the nNOS flavoprotein

Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp(1393)) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (相似文献   

Intramolecular proton-transfer reactions in a membrane-bound proton pump: the effect of pH on the peroxy to ferryl transition in cytochrome c oxidase

In the membrane-bound redox-driven proton pump cytochrome c oxidase, electron- and proton-transfer reactions must be coupled, which requires controlled modulation of the kinetic and/or thermodynamic properties of proton-transfer reactions through the membrane-spanning part of the protein. In this study we have investigated proton-transfer reactions through a pathway that is used for the transfer of both substrate and pumped protons in cytochrome c oxidase from Rhodobacter sphaeroides. Specifically, we focus on the formation of the so-called F intermediate, which is rate limited by an internal proton-transfer reaction from a possible branching point in the pathway, at a glutamic-acid residue (E(I-286)), to the binuclear center. We have also studied the reprotonation of E(I-286) from the bulk solution. Evaluation of the data in terms of a model presented in this work gives a rate of internal proton transfer from E(I-286) to the proton acceptor at the catalytic site of 1.1 x 10(4) s(-1). The apparent pK(a) of the donor (E(I-286)), determined from the pH dependence of the F-formation kinetics, was found to be 9.4, while the pK(a) of the proton acceptor at the catalytic site is likely to be > or = 2.5 pH units higher. In the pH range up to pH 10 the proton equilibrium between the bulk solution and E(I-286) was much faster than 10(4) s(-1), while in the pH range above pH 10 the proton uptake from solution is rate limiting for the overall reaction. The apparent second-order rate constant for proton transfer from the bulk solution to E(I-286) is >10(13) M(-1) s(-1), which indicates that the proton uptake is assisted by a local buffer consisting of protonatable residues at the protein surface.     

Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase

Cytochrome c oxidase couples electron transfer from cytochrome c to O 2 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with O 2, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.     

Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis

Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8. 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex. The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.     

Redox-driven proton pumping by heme-copper oxidases

One of the key problems of molecular bioenergetics is the understanding of the function of redox-driven proton pumps on a molecular level. One such class of proton pumps are the heme-copper oxidases. These enzymes are integral membrane proteins in which proton translocation across the membrane is driven by electron transfer from a low-potential donor, such as, e.g. cytochrome c, to a high-potential acceptor, O(2). Proton pumping is associated with distinct exergonic reaction steps that involve gradual reduction of oxygen to water. During the process of O(2) reduction, unprotonated high pK(a) proton acceptors are created at the catalytic site. Initially, these proton acceptors become protonated as a result of intramolecular proton transfer from a residue(s) located in the membrane-spanning part of the enzyme, but removed from the catalytic site. This residue is then reprotonated from the bulk solution. In cytochrome c oxidase from Rhodobacter sphaeroides, the proton is initially transferred from a glutamate, E(I-286), which has an apparent pK(a) of 9.4. According to a recently published structure of the enzyme, the deprotonation of E(I-286) is likely to result in minor structural changes that propagate to protonatable groups on the proton output (positive) side of the protein. We propose that in this way, the free energy available from the O(2) reduction is conserved during the proton transfer. On the basis of the observation of these structural changes, a possible proton-pumping model is presented in this paper. Initially, the structural changes associated with deprotonation of E(I-286) result in the transfer of a proton to an acceptor for pumped protons from the input (negative) side of the membrane. After reprotonation of E(I-286) this acceptor releases a proton to the output side of the membrane.     

Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (-)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O(2) consumption can be used to compare the rate and functional pK(a) of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (-) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake.     

Estimated acid dissociation constants of the Schiff base, Asp-85, and Arg-82 during the bacteriorhodopsin photocycle.

The pK(a) values of D85 in the wild-type and R82Q, as well as R82A recombinant bacteriorhodopsins, and the Schiff base in the D85N, D85T, and D85N/R82Q proteins, have been determined by spectroscopic titrations in the dark. They are used to estimate the coulombic interaction energies and the pK(a) values of the Schiff base, D85, and R82 during proton transfer from the Schiff base to D85, and the subsequent proton release to the bulk in the initial part of the photocycle. The pK(a) of the Schiff base before photoexcitation is calculated to be in effect only 5.3-5.7 pH units higher than that of D85; overcoming this to allow proton transfer to D85 requires about two thirds of the estimated excess free energy retained after absorption of a photon. The proton release on the extracellular surface is from an unidentified residue whose pK(a) is lowered to about 6 after deprotonation of the Schiff base (Zimanyi, L., G. Varo, M. Chang, B. Ni, R. Needleman, and J.K. Lanyi, 1992. Biochemistry. 31:8535-8543). We calculate that the pK(a) of the R82 is 13.8 before photoexcitation, and it is lowered after proton exchange between the Schiff base and D85 only by 1.5-2.3 pH units. Therefore, coulombic interactions alone do not appear to change the pK(a) of R82 as much and D85 only by 1.5-2.3 pH units. Therefore, coulombic interactions alone do not appear to change the pK(a) of R82 as much as required if it were the proton release group.     

A role for the protein in internal electron transfer to the catalytic center of cytochrome c oxidase

Internal electron transfer (ET) to heme a(3) during anaerobic reduction of oxidized bovine heart cytochrome c oxidase (CcO) was studied under conditions where heme a and Cu(A) were fully reduced by excess hexaamineruthenium. The data show that ET to heme a(3) is controlled by the state of ionization of a single protolytic residue with a pK(a) of 6.5 +/- 0.2. On the basis of the view that ET to the catalytic site is limited by coupled proton transfer, this pK(a) was attributed to Glu60 which is located at the entrance of the proton-conducting K channel on the matrix side of CcO. It is proposed that Glu60 controls proton entry into the channel. However, even with this channel open, there is the second factor that regulates ET, and this is ascribed to the rate of proton diffusion in the channel. In addition, it is concluded that proton transfer in the K channel is reversibly inhibited by the detergent Triton X-100. It is also found that the rate of ET to heme a(3) in the as-isolated resting enzyme and in CcO "activated" by reaction of fully reduced enzyme with O(2) is the same, implying that the catalytic sites of these two forms of oxidized enzyme are essentially identical.     

A Possible Role of Slips in Cytochrome C Oxidase in the Antioxygen Defense System of the Cell

Evidence is available showing that the coupling efficiency of the proton pump in cytochrome c oxidase of mitochondria can under certain conditions decrease significantly below the maximum attainable value. The view is developed that slips in the proton pump of cytochrome c oxidase represent an intrinsic switch mechanism which regulates the relative contribution of energy transfer and respiratory protection against oxygen toxicity by the oxidase.     

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.