List of Publications

1. Curriculum vitae

Name: Vitaliy B. Borisov

Born: October 27, 1969, Tambov, Russia

Place of Work: Dept. Molecular Energetics of Microorganisms, A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia;

Position: Ph.D., Research Scientist in A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University

Academic background:

9/1986 - 7/1992 M. Sc. Biology, first-class diploma

Dept. Biochemistry, School of Biology, Moscow State University

11/1992 - 11/1995 Graduate Student

11/1995 - Ph.D. degree in Biochemistry

11/1995 - 12/1999 - Junior Research Fellow

12/1999 - Present - Research Scientist

A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University

Honours:

04.1999 - the State Stipend for talented young scientists

09.1999 - the State Prize Award for young scientists (the highest scientific award in Russia)

06.2000 - the Award of the Biochemical Society of Russia for young scientists

11.2000 - the Award for young scientists for the best research in A.N.Belozersky Institute

12.2000 - the Award of the Russian Higher Education Academy of Sciences for young scientists

07.2001 - the Award for young scientists for the best research in Moscow State University

01.2002 - the Stipend of Moscow State University for talented young lecturers and scientists

2. List of publications of Dr. Vitaliy B. Borisov

List of full papers

1. Borisov V.B., Smirnova I.A., Krasnosel`skaya I.A., Konstantinov A.A. Oxygenated cytochrome bd from Escherichia coli can be converted into the oxidized form by lipophilic electron acceptors. Biochemistry (Moscow), 1994, v.59, p.437-443.

2. Borisov V.B., Gennis R.B., Konstantinov A.A. Interaction of cytochrome bd from Escherichia coli with hydrogen peroxide. Biochemistry (Moscow), 1995, v.60, p.231-239.

3. Borisov V., Gennis R., Konstantinov A.A. Peroxide complex of cytochrome bd: kinetics of generation and stability. Biochem. Mol. Biol. Int., 1995, v.37, p.975-982.

4. Borisov V.B. Cytochrome bd: Structure and properties. Biochemistry (Moscow), 1996, v.61, p.565-574.

5. Azarkina N., Borisov V., Konstantinov A.A. Spontaneous spectral changes of the reduced cytochrome bd. FEBS Lett., 1997, v.416, p.171-174.

6. Borisov V., Arutyunyan A.M., Osborne J.P., Gennis R.B., Konstantinov A.A. Magnetic circular dichroism used to examine the interaction of Escherichia coli cytochrome bd with ligands. Biochemistry, 1999, v.38, p.740-750.

7. Azarkina N., Siletsky S., Borisov V., Wachenfeldt C., Hederstedt L., Konstantinov A.A. A cytochrome bb’-type quinol oxidase in Bacillus subtilis strain 168. J. Biol. Chem., 1999, v.274, p.32810-32817.

8. Vos M.H., Borisov V.B., Liebl U., Martin J.-L., Konstantinov A.A. Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: a di-heme active site? Proc. Natl. Acad. Sci. USA, 2000, v.97, p.1554-1559.

9. Jasaitis A., Borisov V.B., Belevich N.P., Morgan J.E., Konstantinov A.A., Verkhovsky M.I. Electrogenic reactions of cytochrome bd. Biochemistry, 2000, v.39, p.13800-13809.

10. Borisov V.B., Sedelnikova S.E., Poole R.K., Konstantinov A.A. Interaction of cytochrome bd with carbon monoxide at low and room temperatures. Evidence that only a small fraction of heme b₅₉₅ reacts with CO. J. Biol. Chem., 2001, v.276, p.22095-22099.

11. Borisov V.B., Liebl U., Rappaport F., Martin J.-L., Zhang J., Gennis R.B., Konstantinov A.A., Vos M.H. Interactions between heme d and heme b₅₉₅ in quinol oxidase bd from Escherichia coli: a femtosecond photoselection study. Biochemistry, 2002, v.41, p.1654-1662.

12. Borisov V.B. Defects in mitochondrial respiratory complexes III and IV, and human pathologies. Mol. Aspects Med., 2002, in press.

List of abstracts

1. Borisov V.B., Smirnova I.A., Arutjunjan A.M., Gennis R.B., Konstantinov A.A. Membrane environment effect on ligand-binding properties of E.coli cytochrome bd. Abstract book of the 23rd FEBS Meeting, August 13-18, 1995, Basel, p.241.

2. Borisov V.B., Osborne J.P., Arutjunjan A.M., Smirnova I.A., Gennis R.B., Konstantinov A.A. Interaction of carbon monoxide with cytochrome bd complex from E.coli. Biochim. Biophys. Acta, EBEC Short Reports, 1996, v.9, p.80.

3. Borisov V.B., Osborne J.P., Siletsky S.A., Gennis R.B., Konstantinov A.A. Interaction of the reduced cytochrome bd from E.coli with cyanide. Eur. Biophys. J., 2^nd European Biophysics Congress, 1997, v.26, p.99.

4. Borisov V.B., Osborne J.P., Arutjunjan A.M., Gennis R.B., Konstantinov A.A. Reaction of cytochrome bd oxidase from E.coli with the ligands. Biochim. Biophys. Acta, EBEC Short Reports, 1998, v.10, p.85.

5. Borisov V.B., Osborne J.P., Siletsky S.A., Gennis R.B., Konstantinov A.A. Cyanide complex of the fully reduced cytochrome bd. Biochimie, Abstracts of the 26th FEBS Meeting, June 19-24, 1999, Nice, p.s345.

6. Borisov V.B., Arutyunyan A.M., Konstantinov A.A. Arrangement and function of bd-type terminal oxidases of bacterial respiratory chain. International conference “Mitochondria, cells, and active forms of oxygen”. Puschino, 6-9 June, 2000, p.21-22.

7. Borisov V.B., Vos M.H., Martin J.-L., Konstantinov A.A. Heme-heme interactions in cytochrome bd oxidase studied by femtosecond spectroscopy. Abstract book of the 18th IUBMB/FEBS International Congress of Biochemistry and Molecular Biology, July 16-20, 2000, Birmingham, UK, p. 436.

8. Borisov V.B., Sedelnikova S.E., Poole R.K., Konstantinov A.A. Studies on interaction of cytochrome bd from Azotobacter vinelandii with carbon monoxide at room and low temperatures. Biochim. Biophys. Acta, EBEC Short Reports, 2000, v.11, p.224.

9. Borisov V.B., Jasaitis A., Konstantinov A.A., Belevich N.P., Morgan J.E., Verkhovsky M.I. Flash-induced membrane potential generation by cytochrome bd complex from Escherichia coli. Programme and Abstracts of the FEMS Meeting on the physiology, regulation and biochemistry of electron transfer in microbial catabolism, April 8-12, 2001, Isle of Terschelling, p.101.

10. Borisov V.B., Jasaitis A., Konstantinov A.A., Belevich N.P., Morgan J.E., Verkhovsky M.I. Time-resolved generation of membrane potential by the bd-type quinol oxidase from E.coli. Eur. J. Biochem. Abstracts of the 27th FEBS Meeting, 30 June - 5 July 2001, Lisbon, p.222.

Scientific secretary of

A.N.Belozersky Institute of

Physico-Chemical Biology,

Moscow State University Dr. Z.G. Fetisova

3. List of 5 publications of Dr. Vitaliy B. Borisov with the highest values of impact-factor

1. Azarkina N., Siletsky S., Borisov V., Wachenfeldt C., Hederstedt L., Konstantinov A.A. A cytochrome bb’-type quinol oxidase in Bacillus subtilis strain 168. J. Biol. Chem., 1999, v.274, p.32810-32817.

2. Vos M.H., Borisov V.B., Liebl U., Martin J.-L., Konstantinov A.A. Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: a di-heme active site? Proc. Natl. Acad. Sci. USA, 2000, v.97, p.1554-1559.

3. Jasaitis A., Borisov V.B., Belevich N.P., Morgan J.E., Konstantinov A.A., Verkhovsky M.I. Electrogenic reactions of cytochrome bd. Biochemistry, 2000, v.39, p.13800-13809.

4. Borisov V.B., Sedelnikova S.E., Poole R.K., Konstantinov A.A. Interaction of cytochrome bd with carbon monoxide at low and room temperatures. Evidence that only a small fraction of heme b₅₉₅ reacts with CO. J. Biol. Chem., 2001, v.276, p.22095-22099.

5. Borisov V.B., Liebl U., Rappaport F., Martin J.-L., Zhang J., Gennis R.B., Konstantinov A.A., Vos M.H. Interactions between heme d and heme b₅₉₅ in quinol oxidase bd from Escherichia coli: a femtosecond photoselection study. Biochemistry, 2002, v.41, p.1654-1662.

4. Annotation to the work of Borisov Vitaliy Borisovich “Molecular mechanism of bd-type terminal oxidases functioning”

The work of Dr. V.B.Borisov is devoted to an important problem of biochemistry and molecular bioenergetics - investigation of a mechanism of energy transformation in bacteria on the molecular level. Of particular interest is a structure and a mechanism of function of a bd-type terminal oxidase.

As a terminal ubiquinol-oxidoreductase of a respiratory chain of microorganisms, cytochrome bd catalyzes sequential reduction of oxygen by 4 electrons to 2 molecules of water. Cytochrome bd shows no apparent homology to other known respiratory chain oxidases, such as cytochrome c oxidase, or bo₃-type quinol oxidase. Furthermore, in contrast to other respiratory oxidases, the bd cytochrome complex does not contain copper and does not pump protons across the membrane.

A bd-type terminal oxidase is a key energy-producing respiratory enzyme both in harmless Escherichia coli and in bacteria responsible for different infectious diseases such as dysentery, pneumonia and salmonellosis.

The enzyme consists of two subunits that carry three metal redox centers: low-spin heme b-558 and two high-spin hemes b-595 and d. Heme b-558 is probably the electron acceptor for quinol. When in the ferrous state, heme d anchors O₂ forming a very stable oxycomplex; this heme is likely to be the site at which reduction of oxygen to water takes place. The role of heme b-595 remains to be not clear. It could transfer electron from heme b-558 to heme d or form a second oxygen reactive center or cooperate with heme d to form a ‘binuclear’ oxygen reducing center analogous to heme/copper center of other oxidases.

The purpose of this work was to study an arrangement and function of cytochrome bd oxygen-reducing center. We needed to know how many ligand-binding sites are per an enzyme molecule. In particular, it was necessary to establish, whether high-spin heme b-595 binds external ligands and whether this heme interacts with heme d during sequential reduction of molecular oxygen to water. We aimed at determination of a number of peroxide intermediates formed upon reaction of H₂O₂ with the enzyme. In the course of studies on interaction of cytochrome bd with exogenous ligands, femtosecond multicolor transient absorption spectroscopy, absorption and magnetic circular dichroism (MCD) spectroscopy, and stopped-flow measurements were used.

It was found that interaction of H₂O₂ with cytochrome bd most probably gave a single final product - oxoferryl form of heme d, analogous to intermediate “F” of heme-copper oxidases, whereas the apparent appearance of two distinct spectral intermediates described in the earlier works, could rather originate in the initial presence of two spectrally different forms of heme d (oxidized and oxygenated). MCD signals of cytochrome bd point out binding of CO, NO and cyanide to heme d, however they cannot reveal reaction of these ligands to a major part of high-spin heme b-595. At the same time, at high concentration, these ligands can react with low-spin heme b-558. The participation of cytochrome b-558 in reactions with ligands may complicate significantly interpretation of EPR and other spectroscopy data previously reported. Interaction of the two high-spin hemes in the oxygen reduction site of E.coli cytochrome bd was studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b-595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the a-band of heme b-595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b-595 within a few ps, pointing to a direct interaction between hemes b-595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b-595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b-595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d and heme b-595 on the picosecond time scale.

These results indicate physical proximity of the hemes d and b-595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd. A model of an arrangement and functioning of bd-type terminal oxidases is suggested.

5. Personal contribution of the applicant

The following is to confirm that Dr. Vitaliy B. Borisov was directly involved in planing and carrying out of the experiments, theoretical discussions and preparing of the manuscripts for publication of acquired results. The personal contribution of overall scientific work of the applicant is significant.

A. Konstantinov

6. Abstracts of the papers applied for Academia Europaea Prize for Young Russian Scientists

The cytochrome bd complex as isolated from Escherichia coli under aerobic conditions is in a stable oxygenated form characterized by an intense peak at 650 nm in an absolute absorption spectrum. The commonly used oxidants ferricyanide and persulfate have no effect on the oxygenated form, whereas the addition of lipophilic electron acceptors, such as tetrachlorobenzoquinone or ferricinium, results in the decay of the heme d oxy-complex and the enzyme transition into the fully oxidized form. Interaction of the oxygenated cytochrome bd complex with both tetrachlorobenzoquinone and ferricinium is suppressed by pentachlorophenol, an inhibitor of the enzyme ubiquinol oxidase activity. It is suggested that redox centers of cytochrome bd reside in the hydrophobic environment which can prevent their interaction with the hydrophilic oxidants.

2. Borisov V.B., Gennis R.B., Konstantinov A.A. Interaction of cytochrome bd from Escherichia coli with hydrogen peroxide. Biochemistry (Moscow), 1995, v.60, p.231-239.

The absorption spectrum of the cytochrome bd complex from Escherichia coli in the "as isolated" state is characterized by an intense band at approximately 648 nm belonging to reduced heme d oxycomplex (d²⁺-O₂). This band is often accompanied by a small shoulder around 680 nm. Treatment of the oxycomplex with hydrogen peroxide results in the loss of the 648 nm band and increased absorbance at 680 nm. The peak at 680 nm also appears in the difference absorption spectrum after addition of hydrogen peroxide to the oxidized form of the enzyme and can be attributed to formation of a peroxy or an oxoferryl complex of heme d. The increase in extinction at 680 nm is accompanied by a small red shift of the Soret band; the corresponding difference spectrum with l_min = 405-410 nm and l_max = 430-440 nm is of a magnitude similar to the changes in the visible region (DA_440-410 approximately equals 10 mM^-1cm^-1). This circumstance favours H₂O₂ interaction with heme d rather than b-595. The lineshape of the H₂O₂-induced spectral changes does not vary throughout the hydrogen peroxide concentration range studied (5 mM-5 mM). The H₂O₂ concentration dependence on the 680 nm peak magnitude follows a saturation curve with apparent K_d of 30-40 mM. The product of cytochrome bd interaction with H₂O₂ reacts with cyanide approximately tenfold slower than the free oxidized enzyme. Addition of excess catalase to the hydrogen peroxide-treated cytochrome bd complex fully reverses the H₂O₂-induced spectral changes. However, the rate of disappearance of these changes (k_eff approximately equals 10^-3 s^-1) is ca. 10-fold slower than expected for the dissociation rate constant, k_off, for the peroxy adduct, assuming reversible H₂O₂ binding with K_d approximately equal to 30-40 mM and k_on > 500 M^-1s^-1. This may point to H₂O₂ interaction with cytochrome bd, being essentially irreversible. The initial addition of H₂O₂ to heme d is likely to be followed by cleavage of the O-O bond, giving rise to the oxoferryl state (Fe⁴⁺=O) of heme d which disappears upon removal of H₂O₂ by catalase due to reduction by endogenous electron sources.

3. Borisov V., Gennis R., Konstantinov A.A. Peroxide complex of cytochrome bd: kinetics of generation and stability. Biochem. Mol. Biol. Int., 1995, v.37, p.975-982.

Hydrogen peroxide reacts with the isolated fully oxidized cytochrome bd from Escherichia coli bringing about spectral changes characterized by increased absorption at 680 nm, disappearance of a charge transfer band at 740 nm and a red shift in the Soret band. Only one type of spectral changes is observed throughout the entire range of H₂O₂ concentration studied, 5 - 5000 mM. The absorption changes are consistent with peroxide binding to heme d and do not show any evidence for reaction with heme b-595. The spectral response saturates at increased H₂O₂ concentration with apparent K_d of 30 mM and is reversed by catalase. Stopped-flow measurements show the reaction to be first order with respect to H₂O₂ with a second order rate constant k_on = 600M^-1s^-1. Decay of the H₂O₂-induced spectral changes upon addition of catalase (k approximately 0.001 s^-1) is about 20-fold slower than expected for dissociation of peroxide from the complex with heme d assuming a simple reversible binding of H₂O₂ with K_d and k_on values give above (k_off= K_d*k_on). We suggest that the reaction of H₂O₂ with cytochrome bd may be in fact irreversible, the initial binding followed by a cleavage of the O-O bond and formation of the oxoferryl complex of heme d. Upon removal of excess peroxide, the oxoferryl compound could decay being reduced to the ferric state by endogenous reductants.

4. Borisov V.B. Cytochrome bd: Structure and properties. Biochemistry (Moscow), 1996, v.61, p.565-574.

Literary evidence concerning the arrangement and functioning of the cytochrome bd complex is reviewed with particular emphasis on ligand-binding properties of the enzyme. Some novel data on cytochrome bd interaction with carbon monoxide, cyanide and hydrogen peroxide are presented.

5. Azarkina N., Borisov V., Konstantinov A.A. Spontaneous spectral changes of the reduced cytochrome bd. FEBS Lett., 1997, v.416, p.171-174.

Reduction of the membrane-bound cytochrome bd from Bacillus subtilis, Escherichia coli and Azotobacter vinelandii as well as of the purified enzyme from E. coli was followed by secondary absorption changes on a time scale of tens of minutes. The difference absorption spectra of these changes resembled those induced by CO binding with heme d²⁺ indicating interaction of the heme with an endogenous p-acceptor ligand. The spontaneous spectral changes were prevented and reversed by CO binding with the reduced cytochrome bd. Bonding of heme d iron to an endogenous protein ligand at the sixth axial position upon reduction is proposed and several possible mechanisms of such a process are considered.

The interactions of the fully reduced and fully oxidized cytochrome bd from E. coli with ligands CO, NO, and CN^- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy. In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b-595 and the low-spin heme b-558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component. The data show that at low concentrations, the ligands bind almost exclusively to heme d. At high concentrations, the ligands begin to interact with the low-spin heme b-558. At the same time, no evidence for significant binding of the ligands to the high-spin heme b-595 is revealed in either the reduced or the fully oxidized cytochrome bd complex. The data support the model [Borisov, V. B., Gennis, R. B., and Konstantinov, A. A. (1995) Biochemistry (Moscow) 60, 231-239] according to which the two high-spin hemes d and b-595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b-595.

7. Azarkina N., Siletsky S., Borisov V., Wachenfeldt C., Hederstedt L., Konstantinov A.A. A cytochrome bb’-type quinol oxidase in Bacillus subtilis strain 168. J. Biol. Chem., 1999, v.274, p.32810-32817.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa₃, which is a cytochrome c oxidase, and cytochrome aa₃ 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 aa₃ and caa₃ 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 b₅₅₈b₅₉₅b'.

8. Vos M.H., Borisov V.B., Liebl U., Martin J.-L., Konstantinov A.A. Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: a di-heme active site? Proc. Natl. Acad. Sci. USA, 2000, v.97, p.1554-1559.

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b-595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the a-band of heme b-595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b-595 within a few ps, pointing to a direct interaction between hemes b-595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b-595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b-595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b-595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

9. Jasaitis A., Borisov V.B., Belevich N.P., Morgan J.E., Konstantinov A.A., Verkhovsky M.I. Electrogenic reactions of cytochrome bd. Biochemistry, 2000, v.39, p.13800-13809.

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O₂ and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (Dy, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 ms that matches the formation of ferryl-oxo heme d, the product of the reaction of O₂ with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H₂O₂ with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a Dy of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b-558 and backward. The amplitude of the Dy produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O₂ and is apparently associated with full transmembrane translocation of approximately one charge.

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b-595 with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b-595. However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b-595 is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b-595 and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b-595 in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b-595 for CO is about 20-fold lower than that of heme d.

Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O₂-liganded quinol oxidase bd from Escherichia coli. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b-595 on the picosecond time scale; this general result is fully consistent with previous work [Vos, M. H., Borisov, V. B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b-595 allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b-595 transitions. This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible. CO binding/dissociation from heme d is shown to perturb ferrous heme b-595, causing induction/loss of an absorption band centered at 435 nm. In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b-595 relative to the static spectrum. No evidence for transient binding of CO to heme b-595 after dissociation from heme d is found in the picosecond time range. The yield of CO photodissociation from heme d on a time scale of < 15 ps is found to be diminished more than 3-fold when heme b-595 is oxidized rather than reduced. In contrast to other known heme proteins, molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd at all, indicating a unique structural and electronic configuration of the diheme active site in the enzyme.

12. Borisov V.B. Defects in mitochondrial respiratory complexes III and IV, and human pathologies. Mol. Aspects Med., 2002, in press.

Relationships between alterations in tissue-specific content, protein structure, activity, or/and assembly of respiratory Complexes III and IV induced by mutations in corresponding genes and various human pathologies are reviewed. Cytochrome bc₁ complex and cytochrome c oxidase (COX) deficiencies have been detected in a heterogeneous group of neuromuscular and non-neuromuscular diseases in childhood and adulthood, presenting a number of clinical phenotypes of variableseverity. Such disorders can be caused by mutations located either in mitochondrial genes or in nuclear genes encoding structural subunits of the Complexes or corresponding assembly factors/chaperones. Of the defects in mitochondrial DNA genes, mutations in cytochrome b subunit of Complex III, and in structural subunits I-III of COX have been described to date. As to defects in nuclear DNA genes, mutations in genes encoding the Complexes assembly factors such as the BCS1L protein for Complex III; and SURF-1, SCO1, SCO2, and COX10 for Complex IV have been identified so far.