Vitaliy B. Borisov

The cytochrome bd respiratory oxygen reductases

Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress

Cytochrome bd is a prokaryotic respiratory quinol:O2 oxidoreductase, phylogenetically unrelated to the extensively studied heme-copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O2, NO and H2O2 is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Aerobic respiratory chain of Escherichia coli is not allowed to work in fully uncoupled mode

Escherichia coli is known to couple aerobic respiratory catabolism to ATP synthesis by virtue of the primary generators of the proton motive force—NADH dehydrogenase I, cytochrome bo3, and cytochrome bd-I. An E. coli mutant deficient in NADH dehydrogenase I, bo3 and bd-I can, nevertheless, grow aerobically on nonfermentable substrates, although its sole terminal oxidase cytochrome bd-II has been reported to be nonelectrogenic. In the current work, the ability of cytochrome bd-II to generate a proton motive force is reexamined. Absorption and fluorescence spectroscopy and oxygen pulse methods show that in the steady-state, cytochrome bd-II does generate a proton motive force with a H+/e- ratio of 0.94 ± 0.18. This proton motive force is sufficient to drive ATP synthesis and transport of nutrients. Microsecond time-resolved, single-turnover electrometry shows that the molecular mechanism of generating the proton motive force is identical to that in cytochrome bd-I. The ability to induce cytochrome bd-II biosynthesis allows E. coli to remain energetically competent under a variety of environmental conditions.

Interaction of the bacterial terminal oxidase cytochrome bd with nitric oxide

Cytochrome bd is a prokaryotic terminal oxidase catalyzing O2 reduction to H2O. The oxygen‐reducing site has been proposed to contain two hemes, d and b 595, the latter presumably replacing functionally CuB of heme‐copper oxidases. We show that NO, in competition with O2, rapidly and potently (K i=100±34 nM at ∼70 μM O2) inhibits cytochrome bd isolated from Escherichia coli and Azotobacter vinelandii in turnover, inhibition being quickly and fully reverted upon NO depletion. Under anaerobic reducing conditions, neither of the two enzymes reveals NO reductase activity, which is proposed to be associated with CuB in heme‐copper oxidases.

Cytochrome bd oxidase and nitric oxide: From reaction mechanisms to bacterial physiology

Experimental evidence suggests that the prokaryotic respiratory cytochrome bd quinol oxidase is responsible for both bioenergetic functions and bacterial adaptation to different stress conditions. The enzyme, phylogenetically unrelated to the extensively studied heme–copper terminal oxidases, is found in many commensal and pathogenic bacteria. Here, we review current knowledge on the catalytic intermediates of cytochrome bd and their reactivity towards nitric oxide (NO). Available information is discussed in the light of the hypothesis that, owing to its high NO dissociation rate, cytochrome bd confers resistance to NO‐stress, thereby providing a strategy for bacterial pathogens to evade the NO‐mediated host immune attack.

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O2 to the fully reduced enzyme is followed by the fast (5 μs) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b558 remains reduced while high spin heme b595 is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O2 by two electrons is sufficient to produce (hydro)peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe 3+d-O-O-(H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the PM intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b558 in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b595 are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 μs; it decays into oxoferryl species due to oxidation of low spin heme b558 that is linked to significant charge translocation across the membrane.

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa 3, which is a cytochromec oxidase, and cytochrome aa 3 andbd, 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 andcaa 3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bdcontent 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 andythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion ofythAB in strain LUH27 or the presence of theyth genes on plasmid did not affect the expression of thebb′ oxidase. It is concluded that the novelbb′-type oxidase probably is cytochrome bdencoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e.cytochromeb 558 b 595 b′.

Oxygenated complex of cytochrome bd from Escherichia coli: Stability and photolability

Cytochrome bd is one of the two terminal ubiquinol oxidases in the respiratory chain of Escherichia coli catalyzing reduction of O2 to H2O. The enzyme is expressed under low oxygen tension; due to high affinity for O2 it is isolated mainly as a stable oxygenated complex. Direct measurement of O2 binding to heme d in the one‐electron reduced isolated enzyme gives K d(O2) of ∼280 nM. It is possible to photolyse the heme d oxy‐complex by illumination of the enzyme for several minutes under microaerobic conditions; the light‐induced difference absorption spectrum is virtually identical to the inverted spectrum of O2 binding to heme d.

Cytochrome bd oxidase from Escherichia coli displays high catalase activity: An additional defense against oxidative stress

Cytochrome bd oxygen reductase from Escherichia coli has three hemes, b 558, b 595 and d. We found that the enzyme, as‐prepared or in turnover with O2, rapidly decomposes H2O2 with formation of approximately half a mole of O2 per mole of H2O2. Such catalase activity vanishes upon cytochrome bd reduction, does not compete with the oxygen‐reductase activity, is insensitive to NO, CO, antimycin‐A and N‐ethylmaleimide (NEM), but is inhibited by cyanide (K i ∼2.5 μM) and azide. The activity, possibly associated with heme‐b 595, was also observed in catalase‐deficient E. coli cells following cytochrome bd over‐expression suggesting a protective role against oxidative stress in vivo.

Glutamate 107 in subunit I of cytochrome bd from Escherichia coli is part of a transmembrane intraprotein pathway conducting protons from the cytoplasm to the heme b595/heme d active site.

Cytochrome bd is a terminal quinol:O 2 oxidoreductase of the respiratory chain of Escherichia coli. The enzyme generates protonmotive force without proton pumping and contains three hemes, b 558, b 595, and d. A highly conserved glutamic acid residue of transmembrane helix III in subunit I, E107, was suggested to be part of a transmembrane pathway delivering protons from the cytoplasm to the oxygen-reducing site. When E107 is replaced with leucine, the hemes are retained but the ubiquinol-1-oxidase activity is lost. We compared wild-type and E107L mutant enzymes during single turnover using absorption and electrometric techniques with a microsecond time resolution. Both wild-type and E107L mutant cytochromes bd in the fully reduced state bind O 2 rapidly, but the formation of the oxoferryl species in the mutant is dramatically retarded as compared to the wild type. Intraprotein electron redistribution induced by the photolysis of CO bound to ferrous heme d in the one-electron-reduced wild-type enzyme is coupled to the membrane potential generation, whereas the mutant cytochrome bd shows no such potential generation. The E107L mutation also causes decrease of midpoint redox potentials of hemes b 595 and d by 25-30 mV and heme b 558 by approximately 70 mV. There are two protonatable groups redox-linked to hemes b 595 and d in the active site, one of which has been recently identified as E445, whereas the second group remains unknown. Here we propose that E107 is either the second group or a key residue of a proposed proton delivery pathway leading from the cytoplasm toward this second group.