Plasticity of proton pathway structure and water coordination in cytochrome c oxidase.

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp(132)), near the protein surface, and glutamate Glu(286), which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu(286) affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser(197) (S197A or S197D), located approximately 7 A from Glu(286). Although Ser(197) is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of approximately 35% of that of the wild-type CytcO, and the O(2) reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.

Replacing Asn207 by aspartate at the neck of the D channel in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides results in decoupling the proton pump.

Cytochrome oxidase catalyzes the reduction of O2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all of the pumped protons enter the enzyme through the D channel and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing oxidase activity. N139 is one of three asparagines near the entrance of the D channel, where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pKa of E286, a functionally critical residue that is located 20 A away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pKa of E286. This finding reinforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286, which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.

Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase.

Haem-copper oxidases are the last components of the respiratory chains in aerobic organisms. These membrane-bound enzymes energetically couple the electron transfer (eT) reactions associated with reduction of dioxygen to water, to proton pumping across the membrane. Even though the mechanism of proton pumping at the molecular level still remains to be uncovered, recent progress has presented us with the structural features of the pumping machinery and detailed information about the eT and proton-transfer reactions associated with the pumping process.

Redox-coupled proton translocation in biological systems: proton shuttling in cytochrome c oxidase.

In the respiratory chain free energy is conserved by linking the chemical reduction of dioxygen to the electrogenic translocation of protons across a membrane. Cytochrome c oxidase (CcO) is one of the sites where this linkage occurs. Although intensively studied, the molecular mechanism of proton pumping by this enzyme remains unknown. Here, we present data from an investigation of a mutant CcO from Rhodobacter sphaeroides [Asn-139 --> Asp, ND(I-139)] in which proton pumping is completely uncoupled from the catalytic turnover (i.e., reduction of O2). However, in this mutant CcO, the rate by which O2 is reduced to H2O is even slightly higher than that of the wild-type CcO. The data indicate that the disabling of the proton pump is a result of a perturbation of E(I-286), which is located 20 A from N(I-139) and is an internal proton donor to the catalytic site, located in the membrane-spanning part of CcO. The mutation results in raising the effective pKa of E(I-286) by 1.6 pH units. An explanation of how the mutation uncouples catalytic turnover from proton pumping is offered, which suggests a mechanism by which CcO pumps protons.

Water-hydroxide exchange reactions at the catalytic site of heme-copper oxidases.

Membrane-bound heme-copper oxidases catalyze the reduction of O(2) to water. Part of the free energy associated with this process is used to pump protons across the membrane. The O(2) reduction reaction results in formation of high-pK(a) protonatable groups at the catalytic site. The free energy associated with protonation of these groups is used for proton pumping. One of these protonatable groups is OH(-), coordinated to the heme and Cu(B) at the catalytic site. Here we present results from EPR experiments on the Rhodobacter sphaeroides cytochrome c oxidase, which show that at high pH (9) approximately 50% of oxidized heme a(3) is hydroxide-ligated, while at low pH (6.5), no hydroxide is bound to heme a(3). The kinetics of hydroxide binding to heme a(3) were investigated after dissociation of CO from heme a(3) in the enzyme in which the heme a(3)-Cu(B) center was reduced while the remaining redox sites were oxidized. The dissociation of CO results in a decrease of the midpoint potential of heme a(3), which results in electron transfer (tau approximately equal 3 micros) from heme a(3) to heme a in approximately 100% of the enzyme population. At pH >7.5, the electron transfer is followed by proton release from a H(2)O molecule to the bulk solution (tau approximately equal 2 ms at pH 9). This reaction is also associated with absorbance changes of heme a(3), which on the basis of the results from the EPR experiments are attributed to formation of hydroxide-ligated heme a(3). The OH(-) bound to heme a(3) under equilibrium conditions at high pH is also formed transiently after O(2) reduction at low pH. It is proposed that the free energy associated with electron transfer to the binuclear center and protonation of this OH(-) upon reduction of the recently oxidized enzyme provides the driving force for the pumping of one proton.

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.

A mutation in subunit I of cytochrome oxidase from Rhodobacter sphaeroides results in an increase in steady-state activity but completely eliminates proton pumping.

The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.

Inhibition of proton transfer in cytochrome c oxidase by zinc ions: delayed proton uptake during oxygen reduction.

We have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn(2+) the transition from the "peroxy" (P(R)) to the "ferryl" (F) intermediate has a time constant of approximately 100 micros and it is associated with proton transfer from the bulk solution with an intrinsic time constant of <<100 micros, but rate limited by the P(R)-->F transition. While in the presence of 100 microM Zn(2+) the P(R)-->F transition was slowed by a factor of approximately 2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn(2+)) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of approximately 2.5 ms. Thus, the results show that there is one proton available within the enzyme that can be used for oxygen reduction and confirm our previous observation that F can be formed without proton uptake from the bulk solution. No effect of Zn(2+) was observed with a mutant enzyme in which Asp(I-132), at the entry point of the D-pathway, was replaced by its non-protonatable analogue Asn. In addition, no effect of Zn(2+) was observed on the F-->O transition rate when measured in D(2)O, because in D(2)O, the transition is internally slowed to approximately 10 ms, which is already slower than with bound Zn(2+). Together with earlier results showing that both the P(R)-->F and F-->O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn(2+) binds to and blocks the entrance of the D-pathway.

The rate of internal heme-heme electron transfer in cytochrome C oxidase.

Cytochrome c oxidase catalyzes the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of dioxygen to water. The process involves a number of intramolecular electron-transfer reactions, one of which takes place between the two hemes of the enzyme, hemes a and a3, with a rate of approximately 3 x 10(5) s(-1) (tau approximately 3 micros). In a recent report [Verkhovsky et al. (2001) Biochim. Biophys. Acta 1506, 143-146], it was suggested that the 3 x 10(5) s(-1) electron transfer may be controlled by structural rearrangements and that there is an additional electron transfer that is several orders of magnitude faster. In the present study, we have reinvestigated the spectral changes occurring in the nanosecond and microsecond time frames after photolysis of CO from the fully reduced and mixed-valence enzymes. On the basis of the differences between them, we conclude that in the bovine enzyme the microscopic forward and reverse rate constants for the electron-transfer reactions from heme a to heme a3 are not faster than approximately 2 x 10(5) and approximately 1 x 10(5) s(-1), respectively.

Ligand binding reveals protonation events at the active site of cytochrome c oxidase; is the K-pathway used for the transfer of H(+) or OH(-)?

We have investigated the CO-recombination kinetics after flash photolysis of CO from the "half-reduced" cytochrome c oxidase as a function of pH. In addition, the reaction was investigated in mutant enzymes in which Lys(I-362) and Ser(I-299), located approximately in the middle of the K-pathway and near the enzyme surface, respectively, were modified. Laser-flash induced dissociation of CO is followed by rapid internal electron transfer from heme a(3) to a. At pH>7 this electron transfer is associated with proton release to the bulk solution (tau congruent with 1 ms at pH 8). Thus, the CO-recombination kinetics reflects protonation events at the catalytic site. In the wild-type enzyme, below pH approximately 7, the main component in the CO-recombination displayed a rate of approximately 20 s(-1). Above pH approximately 7, a slow CO-recombination component developed with a rate that decreased from approximately 8 s(-1) at pH 8 to approximately 1 s(-1) at pH 10. This slow component was not observed with KM(I-362), while with the SD(I-299)/SG(I-299) mutant enzymes at each pH it was slower than with the wild-type enzyme. The results are interpreted in terms of proton release from H(2)O in the catalytic site after CO dissociation, followed by OH(-) binding to the oxidized heme a(3). The CO-recombination kinetics is proposed to be determined by the protonation rate of OH(-) and not dissociation of OH(-), i.e. the K-pathway transfers protons and not OH(-). With the KM(I-362) mutant enzyme the proton is not released, i.e. OH(-) is not formed. With the SD(I-299)/SG(I-299) mutant enzymes the proton is released, but both the release and uptake are slowed by the mutations. During reaction of the reduced enzyme with O(2), the H(2)O at the binuclear center is most likely involved as a proton donor in the O-O cleavage reaction.