Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase

We have investigated the electron-proton coupling during the peroxy (P(R)) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of Cu(A) is increased by approximately 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of approximately 200. In ED(I-286) proton uptake during P(R)-->F was slowed by a factor of approximately 5, which indicates that E(I-286) is the proton donor to P(R). In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of approximately 2.5 as compared with approximately 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Adelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of approximately 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.     

Aspartate-132 in cytochrome c oxidase from Rhodobacter sphaeroides is involved in a two-step proton transfer during oxo-ferryl formation.

The aspartate-132 in subunit I (D(I-132)) of cytochrome c oxidase from Rhodobacter sphaeroides is located on the cytoplasmic surface of the protein at the entry point of a proton-transfer pathway used for both substrate and pumped protons (D-pathway). Replacement of D(I-132) by its nonprotonatable analogue asparagine (DN(I-132)) has been shown to result in a reduced overall activity of the enzyme and impaired proton pumping. The results from this study show that during oxidation of the fully reduced enzyme the reaction was inhibited after formation of the oxo-ferryl (F) intermediate (tau congruent with 120 microseconds). In contrast to the wild-type enzyme, in the mutant enzyme formation of this intermediate was not associated with proton uptake from solution, which is the reason the DN(I-132) enzyme does not pump protons. The proton needed to form F was presumably taken from a protonatable group in the D-pathway (e.g., E(I-286)), which indicates that in the wild-type enzyme the proton transfer during F formation takes place in two steps: proton transfer from the group in the pathway is followed by faster reprotonation from the bulk solution, through D(I-132). Unlike the wild-type enzyme, in which F formation is coupled to internal electron transfer from CuA to heme a, in the DN(I-132) enzyme this electron transfer was uncoupled from formation of the F intermediate, which presumably is due to the impaired charge-compensating proton uptake from solution. In the presence of arachidonic acid which has been shown to stimulate the turnover activity of the DN(I-132) enzyme (Fetter et al. (1996) FEBS Lett. 393, 155), proton uptake with a time constant of approximately 2 ms was observed. However, no proton uptake associated with formation of F (tau congruent with 120 micros) was observed, which indicates that arachidonic acid can replace the role of D(I-132), but it cannot transfer protons as fast as the Asp. The results from this study show that D(I-132) is crucial for efficient transfer of protons into the enzyme and that in the DN(I-132) mutant enzyme there is a "kinetic barrier" for proton transfer into the D-pathway.     

Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286)

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.     

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.     

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.     

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.     

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 D-channel of cytochrome oxidase: an alternative view

The D-pathway in A-type cytochrome c oxidases conducts protons from a conserved aspartate on the negatively charged N-side of the membrane to a conserved glutamic acid at about the middle of the membrane dielectric. Extensive work in the past has indicated that all four protons pumped across the membrane on reduction of O(2) to water are transferred via the D-pathway, and that it is also responsible for transfer of two out of the four "chemical protons" from the N-side to the binuclear oxygen reduction site to form product water. The function of the D-pathway has been discussed in terms of an apparent pK(a) of the glutamic acid. After reacting fully reduced enzyme with O(2), the rate of formation of the F state of the binuclear heme-copper active site was found to be independent of pH up to pH~9, but to drop off at higher pH with an apparent pK(a) of 9.4, which was attributed to the glutamic acid. Here, we present an alternative view, according to which the pH-dependence is controlled by proton transfer into the aspartate residue at the N-side orifice of the D-pathway. We summarise experimental evidence that favours a proton pump mechanism in which the proton to be pumped is transferred from the glutamic acid to a proton-loading site prior to proton transfer for completion of oxygen reduction chemistry. The mechanism is discussed by which the proton-pumping activity is decoupled from electron transfer by structural alterations of the D-pathway. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

The onset of the deuterium isotope effect in cytochrome c oxidase

We have investigated the dynamics of proton equilibration within the proton-transfer pathway of cytochrome c oxidase from bovine heart that is used for the transfer of both substrate and pumped protons during reaction of the reduced enzyme with oxygen (D-pathway). The kinetics of the slowest phase in the oxidation of the enzyme (the oxo-ferryl --> oxidized transition, F --> O), which is associated with proton uptake, were studied by monitoring absorbance changes at 445 nm. The rate constant of this transition, which is 800 s(-)(1) in H(2)O (at pH approximately 7.5), displayed a kinetic deuterium isotope effect of approximately 4 (i.e., the rate was approximately 200 s(-)(1) in 100% D(2)O). To investigate the kinetics of the onset of the deuterium isotope effect, fully reduced, solubilized CO-bound cytochrome c oxidase in H(2)O was mixed rapidly at a ratio of 1:5 with a D(2)O buffer saturated with oxygen. After a well-defined time period, CO was flashed off using a short laser flash. The time between mixing and flashing off CO was varied within the range 0. 04-10 s. The results show that for the bovine enzyme, the onset of the deuterium isotope effect takes place within two time windows of O transition is internal proton transfer from a site, proposed to be Glu (I-286) (R. sphaeroides amino acid residue numbering), to the binuclear center. The spontaneous equilibration of protons/deuterons with this site in the interior of the protein is slow (approximately 1 s).     

Ligand binding and the catalytic reaction of cytochrome caa(3) from the thermophilic bacterium Rhodothermus marinus

The ligand-binding dynamics and the reaction with O(2) of the fully (five-electron) reduced cytochrome caa(3) from the thermohalophilic bacterium Rhodothermus (R.) marinus were investigated. The enzyme is a proton pump which has all the residues of the proton-transfer pathways found in the mitochondrial-like enzymes conserved, except for one of the key elements of the D-pathway, the helix-VI glutamate [Glu(I-286), R. sphaeroides numbering]. In contrast to what has been suggested previously as general characteristics of thermophilic enzymes, during formation of the R. marinus caa(3)-CO complex, CO binds weakly to Cu(B), and is rapidly (k(Ba) = 450 s(-1)) trapped by irreversible (K(Ba) = 4.5 x 10(3)) binding to heme a(3). Upon reaction of the fully reduced enzyme with O(2), four kinetic phases were resolved during the first 10 ms after initiation of the reaction. On the basis of a comparison to reactions observed with the bovine enzyme, these phases were attributed to the following transitions between intermediates (pH 7.8, 1 mM O(2)): R --> A (tau congruent with 8 micros), A --> P(r) (tau congruent with 35 micros), P(r) --> F (tau congruent with 240 micros), F --> O (tau congruent with 2.5 ms), where the last two phases were associated with proton uptake from the bulk solution. Oxidation of heme c was observed only during the last two reaction steps. The slower transition times as compared to those observed with the bovine enzyme most likely reflect the replacement of Glu(I-286) of the helix-VI motif -XGHPEV- by a tyrosine in the R. marinus enzyme in the motif -YSHPXV-. The presence of an additional, fifth electron in the enzyme was reflected by two additional kinetic phases with time constants of approximately 20 and approximately 720 ms during which the fifth electron reequilibrated within the enzyme.     

The entry point of the K-proton-transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump. The enzyme has two proton input pathways, leading from the solution on the N-side to the binuclear center. One of these pathways, the K-pathway, is used for proton uptake upon reduction of the binuclear center. It is also important for local charge compensation during reaction of the fully reduced enzyme with O2. Two different locations have been proposed to constitute the entry point of the K-pathway: near S(I-299) or near E(II-101), respectively, in the Rhodobacter sphaeroides enzyme. The experiments discussed in this study are aimed at identifying the location of the entry point. The kinetics and extent of flash-induced proton release coupled to oxidation of heme a3 (tau congruent with 2 ms at pH 8.8 in the wild-type enzyme) in the absence of O2 were investigated in the ED(II-101), SD(I-299), and KM(I-362) mutant enzymes, i.e., at the two proposed entry points and in the middle of the pathway, respectively. This reaction was completely blocked in KM(I-362), while it was slowed by factors of 25 and 40 in the ED(II-101) and SD(I-299) mutant enzymes, respectively. During reaction of the fully reduced enzyme with O2, electron transfer from heme a to the catalytic site (during P(R)-formation) was blocked in the KM(I-362) and SD(I-299)/SG(I-299) but not in the ED(II-101)/ EA(II-101) mutant enzymes. The results are interpreted as follows: Residue K(I-362) is involved in both proton transfer and charge compensation (in different reaction steps). The impaired proton release in the S(I-299) mutant enzymes is an indirect effect due to an altered environment of K(I-362). E(II-101), on the other hand, is likely to be part of the K-pathway since mutation of this residue results in impaired proton release but does not affect the P(R) formation kinetics; i.e., the properties of K(I-362) are not altered. Consequently, we conclude that the entry point of the K-pathway is located near E(II-101).     

Calculated proton uptake on anaerobic reduction of cytochrome C oxidase: is the reaction electroneutral?

Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O(2). Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a(3) and Cu(B). If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK(a)s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu(B) shifts its pK(a) from below 7 to above 7 and so picks up a proton when heme a(3) and Cu(B) are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a(3) all have pK(a)s above 7 in all redox states, although they have only 1.6-3.5 DeltapK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK(a)s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu(A), heme a, heme a(3), and Cu(B) shows approximately 2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E(m)) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E(m) of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E(m) is independent of the BNC redox state.     

Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.     

Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes

The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Br?nsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.     

An elementary reaction step of the proton pump is revealed by mutation of tryptophan-164 to phenylalanine in cytochrome c oxidase from Paracoccus denitrificans

Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

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.     

Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides

We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides with a view to evaluating the role of this residue in redox-linked proton translocation. The mutation E286D did not have any dramatic effects on enzyme properties and retained 50% of wild-type catalytic activity. For E286Q a fraction of the binuclear center was trapped in an unreactive, spectrally distinct form which is most likely due to misfolded protein, but the majority of E286Q reacted normally with formate and cyanide in the oxidized state, and with carbon monoxide and cyanide in the dithionite-reduced form. The mutation also had little effect on the pH-dependent redox properties of haem a in the reactive fraction. However, formation of the P state from oxidized enzyme with hydrogen peroxide or by aerobic incubation with carbon monoxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic steady state in the presence of ferrous cytochrome c was characterized by essentially fully reduced haem a and ferric haem a3, suggesting that the mutation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a3 following anaerobiosis, there was no indication of accumulation of significant amounts of P state. We propose that the glutamate is implicated in several steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.     

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.     

Proton stoichiometry in the reduction of the FAD and disulfide of Escherichia coli thioredoxin reductase. Evidence for a base at the active site

The oxidation-reduction midpoint potentials, Em, of the FAD and active site disulfide couples of Escherichia coli thioredoxin reductase have been determined from pH 5.5 to 8.5. The FAD and disulfide couples have similar Em values and thus a linked equilibrium of four microscopic enzyme oxidation-reduction states exists. The binding of phenylmercuric acetate to one enzyme form could be monitored which allowed solving the four microscopic Em values. The Em values at pH 7.0 and 12 degrees C of the four couples of thioredoxin reductase are: (S)2-enzyme-FAD/FADH2 = -0.243 V, (SH)2-enzyme-FAD/FADH2 = -0.260 V, (FAD)-enzyme-(S)2/(SH)2 = -0.254 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.271 V. Thus, at pH 7.0, the FAD and disulfide moieties have a 0.017-V negative interaction and Em values which are different by 0.011 V. The delta Em/delta pH of the FAD couples E2m and E3m are about 0.060 V/pH throughout the pH range studied, showing an approximately 2-proton stoichiometry of reduction of the enzyme FAD. The delta Em/delta pH of the disulfide couples E1m and E4m are about 0.052 V/pH from pH 5.5 to 8.5, showing an apparently nonintegral proton stoichiometry of reduction of 1.8 in this pH range. This proton stoichiometry suggests the presence of a base with an ionization behavior that is linked to the oxidation-reduction state of the disulfide. A novel method is presented for determining the pK values on oxidized and reduced enzyme which agrees with the less accurate classical method. The proton stoichiometry results are consistent with the presence of a thiol-base ion pair in which the pK of the base is elevated from 7.6 in disulfide containing enzyme to greater than 8.5 upon forming an ion pair with a thiol anion of pK 7.0 generated upon reduction of the disulfide. The fluorescence of the FAD in thioredoxin reductase decreases as the pH is lowered with a pK of 7.0, direct evidence for a base near the FAD probably distinct from the base interacting with the dithiol.