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Electrophysiological approaches to the study of the activity of retinal-containing protein bacteriorhodopsin (bR) or other proteins of this family are based usually on measurements of electrical current through a planar bilayer lipid membrane (BLM) with proteoliposomes attached to the BLM surface at one side of the membrane. Here, we describe the measurements of the pumping activity of bR and channelrhodopsin 2 (ChR2) with special attention to the study of voltage dependence of the light-induced currents. Strong voltage dependence of ChR2 suggests light-triggered ion channel activity of ChR2. We also describe electrophysiological measurements with the help of collodion film instead of BLM for the measurements of fast stages of a rhodopsin photocycle as well as the estimation of the activity of proteoliposomes without a macro membrane using fluorescent probes such as oxonol VI or 9-aminoacridine.

Microbial rhodopsins are the family of retinal-containing proteins that perform primarily the light-driven transmembrane ion transport and sensory functions. They are widely distributed in nature and can be used for optogenetic control of the cellular activities by light. Functioning of microbial rhodopsins results in generation of the transmembrane electric potential in response to a flash that can be measured by direct time-resolved electrometry. This method was developed by L. Drachev and his colleagues at the Belozersky Institute and successfully applied in the functional studies of microbial rhodopsins. First measurements were performed using bacteriorhodopsin from

The effect of Zn2+ on the P-side of proteoliposomes containing membrane-incorporated Rhodobacter sphaeroides cytochrome c oxidase was investigated by the time-resolved electrometrics following a single electron injection into the enzyme prepared in the F state. The wild-type enzyme was examined along with the two mutants, N139D and D132N. All obtained data indicate that the primary effect of Zn2+ added from the P-side of the membrane is slowing of the pumped proton release from the proton loading site (PLS) to the bulk aqueous phase on the P-side of the membrane. The results strongly suggest the presence of two pathways by which the pumped proton can exit the protein from the PLS and of two separate binding sites for Zn2+. A model is presented to explain the influence of Zn2+ on the kinetics of membrane-potential generation by the wild-type COX, as well as by the N139D and D132N mutants.

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 &plusmn, 2.7 mM (NH4)2SO4 (4.9 &plusmn, 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 &plusmn, 7.1 and 24.6 &plusmn, 12.4 mM (NH4)2SO4 (7.2 &plusmn, 1.4 and 4.9 &plusmn, 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

Cytochrome ba

Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target.\ud \ud \ud \ud Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed.\ud \ud \ud \ud Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands.\ud \ud \ud \ud Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome bd is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome bd is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.

Terminal respiratory oxidases are highly efficient molecular machines. These most important bioenergetic membrane enzymes transform the energy of chemical bonds released during the transfer of electrons along the respiratory chains of eukaryotes and prokaryotes from cytochromes or quinols to molecular oxygen into a transmembrane proton gradient. They participate in regulatory cascades and physiological anti-stress reactions in multicellular organisms. They also allow microorganisms to adapt to low-oxygen conditions, survive in chemically aggressive environments and acquire antibiotic resistance. To date, three-dimensional structures with atomic resolution of members of all major groups of terminal respiratory oxidases, heme-copper oxidases, and bd-type cytochromes, have been obtained. These groups of enzymes have different origins and a wide range of functional significance in cells. At the same time, all of them are united by a catalytic reaction of four-electron reduction in oxygen into water which proceeds without the formation and release of potentially dangerous ROS from active sites. The review analyzes recent structural and functional studies of oxygen reduction intermediates in the active sites of terminal respiratory oxidases, the features of catalytic cycles, and the properties of the active sites of these enzymes.

Two electrogenic phases with characteristic times of ~ 14 μs and ~ 290 μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized “relaxed” O state of ba 3 oxidase from T. thermophilus (O → E transition). The rapid phase reflects electron redistribution between Cu A and heme b . The slow phase includes electron redistribution from both Cu A and heme b to heme a 3 , and electrogenic proton transfer coupled to reduction of heme a 3 . The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a 3 is reduced, but there is no proton pumping and no reduction of Cu B . Single-electron reduction of the oxidized “unrelaxed” state (O H → E H transition) is accompanied by electrogenic reduction of the heme b /heme a 3 pair by Cu A in a “fast” phase (~ 22 μs) and transfer of protons in “middle” and “slow” electrogenic phases (~ 0.185 ms and ~ 0.78 ms) coupled to electron redistribution from the heme b /heme a 3 pair to the Cu B site. The “middle” and “slow” electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach Cu B the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~ 0.1 H + /e − , probably due to the formed membrane potential in the experiment.

Recombination of the isolated, fully reduced bd-type quinol oxidase from Escherichia coli with carbon monoxide was studied by pulsed absorption spectrophotometry with microsecond time resolution. Analysis of the kinetic phases of recombination was carried out using the global analysis of multiwavelength kinetic data (“Global fitting”). It was found that the unresolved photodissociation of CO is followed by a stepwise (with four phases) recombination with characteristic times (τ) of about 20 μs, 250 μs, 1.1 ms, and 24 ms. The 20-μs phase most likely reflects bimolecular recombination of CO with heme d. Two subsequent kinetic transitions, with τ ~ 250 μs and 1.1 ms, were resolved for the first time. It is assumed that the 250-μs phase is heterogeneous and includes two different processes: recombination of CO with ~7% of heme b595 and transition of heme d from a pentacoordinate to a transient hexacoordinate state in this enzyme population. The 24-ms transition probably reflects a return of heme d to the pentacoordinate state in the same protein fraction. The 1.1-ms phase can be explained by recombination of CO with ~15% of heme b558. Possible models of interaction of CO with different heme centers are discussed.

Terminal oxidases of aerobic respiratory chains catalyze the transfer of electrons from the respiratory substrate, cytochrome c or quinol, to O2 with the formation of two H2O molecules. There are two known families of these membrane oxidoreductases: heme-copper oxidase superfamily and bd-type oxidase family (cytochromes bd) found in prokaryotes only. The redox reaction catalyzed by these enzymes is coupled to the generation of proton motive force used by the cell to synthesize ATP and to perform other useful work. Due to the presence of the proton pump, heme-copper oxidases create the membrane potential with a greater energy efficiency than cytochromes bd. The latter, however, play an important physiological role that enables bacteria, including pathogenic ones, to survive and reproduce under adverse environmental conditions. This review discusses the features of organization and molecular mechanisms of functioning of terminal oxidases from these two families in the light of recent experimental data.

Bacteria can not only encounter carbon monoxide (CO) in their habitats but also produce the gas endogenously. Bacterial respiratory oxidases, thus, represent possible targets for CO. Accordingly, host macrophages were proposed to produce CO and release it into the surrounding microenvironment to sense viable bacteria through a mechanism that in Escherichia (E.) coli was suggested to involve the targeting of a bd-type respiratory oxidase by CO. The aerobic respiratory chain of E. coli possesses three terminal quinol:O

Bacteria can not only encounter carbon monoxide (CO) in their habitats but also produce the gas endogenously. Bacterial respiratory oxidases, thus, represent possible targets for CO. Accordingly, host macrophages were proposed to produce CO and release it into the surrounding microenvironment to sense viable bacteria through a mechanism that in Escherichia (E.) coli was suggested to involve the targeting of a bd-type respiratory oxidase by CO. The aerobic respiratory chain of E. coli possesses three terminal quinol:O2-oxidoreductases: the heme-copper oxidase bo3 and two copper-lacking bd-type oxidases, bd-I and bd-II. Heme-copper and bd-type oxidases differ in the mechanism and efficiency of proton motive force generation and in resistance to oxidative and nitrosative stress, cyanide and hydrogen sulfide. Here, we investigated at varied O2 concentrations the effect of CO gas on the O2 reductase activity of the purified cytochromes bo3, bd-I and bd-II of E. coli. We found that CO, in competition with O2, reversibly inhibits the three enzymes. The inhibition constants Ki for the bo3, bd-I and bd-II oxidases are 2.4 ± 0.3, 0.04 ± 0.01 and 0.2 ± 0.1 μM CO, respectively. Thus, in E. coli, bd-type oxidases are more sensitive to CO inhibition than the heme-copper cytochrome bo3. The possible physiological consequences of this finding are discussed.

Reactive oxygen species (ROS) comprise the superoxide anion (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen (1O2). ROS can damage a variety of macromolecules, including DNA, RNA, proteins, and lipids, and compromise cell viability. To prevent or reduce ROS-induced oxidative stress, bacteria utilize different ROS defense mechanisms, of which ROS scavenging enzymes, such as superoxide dismutases, catalases, and peroxidases, are the best characterized. Recently, evidence has been accumulating that some of the terminal oxidases in bacterial respiratory chains may also play a protective role against ROS. The present review covers this role of terminal oxidases in light of recent findings.

ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H+ transfer.

Two electrogenic phases with characteristic times of ~14μs and ~290μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba

A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.

Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome-reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50-90%, depending on preparation) with an effective K(i) of approximately 10 microM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes, and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme: full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 microM. The rate of zinc inhibitory action in the slow phase is proportional to Zn(2+) concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F --> O step corresponding to transfer of the 4th electron in the catalytic cycle).

The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo --> oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Deltaphi in the ferryl-oxo --> oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (tau approximately 15 micros), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (tau approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl-oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl-oxo-to-oxidized (F --> O) transition of the enzyme [Zaslavsky, D., et al. (1993) FEBS Lett. 336, 389-393]. As in the case of the F --> O transition, the electric response associated with photoreduction of P to F includes a rapid KCN-insensitive electrogenic phase with a tau of 40-50 microseconds (reduction of heme a by CuA) and a multiphasic slower part; this part is cyanide-sensitive and is assigned to vectorial transfer of protons coupled to reduction of oxygen intermediate in the binuclear center. The net KCN-sensitive phase of the response is approximately 4-fold more electrogenic than the rapid phase, which is similar to the characteristics of the F --> O electrogenic transition and is consistent with net transmembrane translocation of two protons per electron, including vectorial movement of both "chemical" and "pumped" protons. The protonic part of the P --> F electric response is faster than in the F --> O transition and can be deconvoluted into three exponential phases with tau values varying for different samples in the range of 0.25-0.33, 1-1.5, and 6-7.5 ms at pH 8. Of these three phases, the 1-1.5 ms component is the major one contributing 50-60%. The P --> F conversion induced by single electron photoreduction of the peroxy state as studied in this work is several times slower than the P --> F transition resolved during oxidation of the fully reduced oxidase by molecular oxygen. The role of the CuB redox state in controlling the rate of P --> F conversion of heme a3 is discussed.

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides . A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a 3 (Fe 4+ = O 2− ) to the oxidized form (Fe 3+ OH − ). This redox step requires the delivery of a “chemical” H + to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a 3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.

The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an approximately 15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the F --> O transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O(2) shows approximately 200-fold inhibition of the F --> O step (k approximately 2 s(-1) at pH 8) which is not compatible with enzyme turnover ( approximately 30 s(-1)). Presumably, an abnormal intermediate F(deprotonated) is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the F --> O transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H(2)O(2), decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Deltapsi generation coupled to the flash-induced F --> O transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a(3) from the Fe(4+) horizontal lineO(2-) state to the Fe(3+)-OH(-) state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the F --> O step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of approximately 10 and approximately 40 mus and a magnitude ratio of approximately 3:2. The 10 mus phase matches vectorial electron transfer from Cu(A) to heme a, whereas the 40 mus component is assigned to intraprotein proton displacement across approximately 20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D-channel mutants as well as with cyanide-inhibited wild-type oxidase. The finding helps to reconcile the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme ( approximately 30%) with the net electrogenicity of the F --> O transition coupled to transmembrane transfer of two charges per electron.

The P(M)--F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P(M) state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is approximately 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F--O transition, but is significantly slower than formation of the F state from the P(R) intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70-90 micros). The approximately 0.3 ms P(M)--F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P(M)--F transition is generated much later, with a time constant of 1.3 ms. In addition, the P(M)--F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with tau values of 0.18 and 0.85 ms.

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q (A) (-) and the oxidized non-heme iron Fe(3+) on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Deltapsi (negative charging of the proteoliposome interior) indicates that the iron-quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (tau approximately 0.1 micro s) of Deltapsi generation related to electron transfer between the redox-active tyrosine Y(Z) and Q(A), an additional phase in the submillisecond time domain (tau approximately 0.1 ms at 23 degrees C, pH 7.0) and relative amplitude approximately 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q(A) and the secondary quinone Q(B). The rate of the additional electrogenic phase is decreased by about one-half in the presence of D(2)O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.

Cytochrome c oxidase (COX) terminates the respiratory chain in mitochondria and in plasmatic membrane of many aerobic bacteria. The enzyme reduces dioxygen molecule into water and the reaction is accompanied with generation of transmembrane difference of electric potentials. The energy conservation by COX is based on the vectorial organization of the chemical reaction due to substrate protons transfer from the negative phase into the active site to meet the electrons, coming from opposite side of the membrane. In addition, a half of free energy of redox-chemistry reaction is conserved through transmembrane proton pumping. Each of the reaction steps in the catalytic cycle of COX involves a sequence of coupled electron and proton transfer reaction. The time-resolved study of the coupled charge translocation during catalytic cycle of cytochrome c oxidase is reviewed. Based on many fascinating parallels between the mechanisms of COX and pigment-protein complex of photosystem II from oxygenic photosynthetic organisms, the data described could be used in the light-driven water-splitting process.

Yeast iso-1-cytochrome c covalently modified at cysteine-102 with (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]Ru2+ (Ru-102-Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash-induced photoreduction of Ru-102-Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipyridyl)3 (RuBpy) as the photoreductant [D.L. Zaslavsky et al. (1993) FEBS Lett. 336, 389-393]. At low ionic strength, when Ru-102-Cyt c forms a tight electrostatic complex with COX, flash-activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles. The initial rapid phase is virtually identical to the 50 microsecond transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by CuA. CuA reduction by Ru-102-Cyt c turns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme. A millisecond phase (tau ca. 4 ms) following the 50 microsecond initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru-102-Cyt c as electron donors; however, this phase is small in the absence of added H2O2. In addition to these two transients, the flash-induced electrogenic response in the presence of Ru-102-Cyt c reveals a large slow phase of delta psi generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 microM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru-102-Cyt c2+ generated by the flash in a solution.

The kinetics of the oxidation of fully-reduced ba3 cytochrome c oxidase from Thermus thermophilus by oxygen were followed by timeresolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa3-type oxidases. However the F intermediate in ba3 oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P →F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F →O transition in ba3 oxidase is close to that observed during the F →O transition in the aa3 oxidases. However, the PR →F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers. © 2007 Elsevier B.V. All rights reserved.

The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa3 cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a3 to heme a and further to the cytochrome c/CuA has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a3 to heme a and further to the cytochrome c/CuA pair is resolved as a single transition with τ ~ 40 μs. In the reaction of the fully reduced cytochrome caa3 with oxygen, the first electrogenic phase (τ ~ 30 μs) is linked to O O bond cleavage and generation of the PR state. The next electrogenic component (τ ~ 50 μs) is associated with the PR → F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ~ 0.25 ms, ~ 1.4 ms and ~ 4 ms, are linked to the conversion of the binuclear center through the F → OH → EH transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.

The kinetics of single-electron injection into the oxidized nonrelaxed state (O H → E H transition) of the aberrant ba 3 cytochrome oxidase from Thermus thermophilus , noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component ( τ ∼ 17 μs) reflects oxidation of Cu A and reduction of the heme groups (low-spin heme b and high-spin heme a 3 in a ratio close to 50:50). The subsequent component ( τ ∼ 420 μs) includes reoxidation of both hemes by Cu B . This is in significant contrast to the O H → E H transition of the aa 3 -type cytochrome oxidase from Paracoccus denitrificans , where the fastest phase is exclusively due to transient reduction of the low-spin heme a , without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba 3 oxidase was similar to that in aa 3 oxidase and only included rapid electron transfer from Cu A to the low-spin heme b . This indicates a functional difference between the relaxed O and the pulsed O H forms also in the ba 3 oxidase from T. thermophilus .