The cytochrome bd respiratory oxygen reductases

Summary

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.

1. Diversity of respiratory oxygen reductases

Respiratory oxygen reductases (terminal oxidases) are enzymes at the end of the respiratory chains of organisms which couple the oxidation of a respiratory substrate (one-electron donor, cytochrome c, or two-electron donor, quinol (QH₂)) to the four-electron reduction of O₂ to water. There are three families of oxygen reductases (Fig. 1).

Fig. 1.

Respiratory oxygen reductases. The bd-family is subdivided into the A-subfamily (long Q-loop), B-subfamily (short Q-loop) and the cyanide insensitive oxygen reductases (CIO). These are subdivisions based entirely on spectroscopic and structural observations and are not phylogentically defined clades.

1.1 Heme-copper family

The first, most extensively studied family comprises the heme-copper oxygen reductases. They have a binuclear O₂-reduction site composed of a high spin heme (a₃, o₃, or b₃) and a copper ion (Cu_B), and these enzymes generate a PMF via a “proton pump” mechanism [1–7]. The PMF is utilized for various biosynthetic activities (e.g., ATP production), solute active transport and mechanical movement (e.g., flagellar rotation). The heme-copper family of oxygen reductases includes both cytochrome c oxidases and quinol oxidases. Most of the heme-copper oxygen reductases are members of one of three distinct subfamilies: A, B, and C [8,9]. The A subfamily includes the mitochondrial cytochrome c oxidases as well as many prokaryotic cytochrome c oxidases and quinol oxidases. Enzymes in the A-subfamily utilize at least two proton pathways to deliver protons to the active site or for proton pumping. The B subfamily includes a number of oxygen reductases from extremophilic prokaryotes, such as the ba₃-type oxygen reductase from T. thermophilus [10]. The enzymes of the C subfamily are all cbb₃-type oxidases [11]. Recently, it has been shown that the enzymes from the B and C subfamilies utilize only one proton-conducting input pathway [10,12]. High-resolution x-ray crystal structures of the heme-copper oxidases from all three subfamilies have been reported [11,13–23].

1.2 Alternative oxidase (AOX) family

The second family of respiratory oxygen reductases comprises cyanide-resistant AOX found in mitochondria of higher plants, fungi and protists as well as in prokaryotes and some animal species [24]. In plants, this is a homodimeric enzyme associated with the matrix side of the inner mitochondrial membrane. AOX uses UQH₂, but not cytochrome c, as the electron donor, and contains a non-heme di-iron carboxylate active site for O₂ reduction.

AOX does not produce a PMF, and is not coupled to transmembrane charge transfer. However, AOX is responsible for heat generation in some tissues, and plays a role in the regulation of energy metabolism, facilitating turnover of the TCA cycle, protection against oxidative stress, and homeostasis. To date, no high-resolution AOX structure has been reported, but crystals that diffract to better than 3.0 Å have been described [25].

1.3 Cytochrome bd-family

The third family of oxygen reductases comprises cytochromes bd. These are quinol oxidases found in a wide variety of prokaryotes. They show no sequence homology to any subunit of heme–copper family members or AOX and do not contain any copper or non-heme iron [26–33]. This two-subunit integral membrane protein (subunits I and II) contains three hemes, b₅₅₈, b₅₉₅ and d, and it is generally thought that hemes b₅₉₅ and d form a di-heme site for the reduction of O₂ (Fig. 2) [34–43]. Unfortunately, no X-ray structure of any bd-type oxygen reductase has been reported. Cytochrome bd generates a PMF by transmembrane charge separation, but does so without being a “proton pump” [41,44–50]. In a number of organisms, the bd oxygen reductase is induced under O₂-limited conditions as well as under other growth conditions that can be considered stressful, such as Fe deficiency [51–54]. All known members of the bd-family of oxygen reductases are quinol oxidases, most commonly using ubiquinol or menaquinol as substrates.

Fig. 2.

Proposed cytochrome bd model.

Analysis of prokaryotic genomes shows that many aerobic prokaryotes do not contain any member of the bd-family, but contain only heme-copper oxygen reductases. However, there are a number of prokaryotes that encode more than one bd-family member, for example, two: E. coli [53,55], Bacillus subtilis [56]; three: Vibrio cholerae [57]; and as many as six bd-type oxygen reductases: some Acidithiobacillus strains. Organisms that express one or more bd-type oxygen reductases, usually also possess at least one heme-copper oxygen reductase. However in some cases (e.g., Lactobacillus plantarum [58], Zymomonas mobilis [59], the two Thermoplasma strains [60]) cytochrome bd is the only oxygen reductase.

1.3.1 The Q-loop

The hydrophilic region of subunit I connecting transmembrane helices 6 and 7, facing the outside of the prokaryotic cell, has been implicated as part of the quinol binding site [61–66], and this is referred to as the “Q loop”. Some of the bd-family oxygen reductases have an insert in the C-terminal portion of the Q-loop and, hence, have a “long Q-loop”, e.g., enzymes isolated from Escherichia coli and Azotobacter vinelandii [67,68]. The majority of bd-type oxygen reductases have a “short Q-loop”, e.g., the enzyme isolated from Bacillus stearothermophilus [67–69]. It is not clear what the functional consequences are, if any, from this difference in the size of the Q loop.

1.3.2 Cyanide insensitive oxidases (CIO)

An anecdotal observation is that some of the “short Q-loop” oxygen reductases appear to have an altered heme content, in which the amount of heme d is significantly reduced (or totally missing) and is replaced by a heme b. This appears to be the case for a B. subtilis cytochrome bd [70]. When these enzymes, with a low content of heme d, have been characterized in bacterial membranes, respiration continues even in the presence of 1 mM KCN [71], but the membranes do not have the spectroscopic signature of heme d (a peak in the reduced form near 630 nm) [71–75]. As a result, these enzymes have been called cyanide insensitive oxidases (CIO) [73]. Examples are P. aeruginosa [71–73,76], P. putida [77], P. pseudoalcaligenes [74], Staphylococcus carnosus [78], C. jejuni [75], Z. mobilis [59]. On the contrary, using low temperature absorption spectroscopy, EPR and mass spectrometry, Mogi et al. [79] reported that CIO in the membranes from G. oxydans has the same heme contents present in a classical cytochrome bd, although reveals unique spectroscopic and ligand-binding properties. Whether the CIO heme composition is strain- and/or growth-specific, or the heme spectral features were not detected due to a very low enzyme concentration in the tested membranes remains to be studied. It is now clear that CIOs are bd-family oxygen reductases.

cioA and cioB genes which encode CIO in P. aeruginosa and P. pseudoalcaligenes were sequenced [73,74]. They comprise the cio operon. CioA and CioB are homologous to subunits I and II of cytochrome bd-I from E. coli and the bd-oxidase from A. vinelandii [73]. Histidine and methionine residues identified in cytochrome bd-I from E. coli as the axial ligands to heme b₅₅₈ and heme b₅₉₅ are conserved [73]. It was proposed that the slight differences in sequence and structure of the CydB subunit are responsible for cyanide resistance [78]. It is of interest to note that cytochrome bd of the cyanobacterium Synechocystis sp. PCC 6803 appeared to be structurally related to CIO [80]. To date, no CIO has been purified and characterized, primarily because these enzymes appear to be particularly labile. At low O₂ tensions, the opportunistic pathogen P. aeruginosa synthesizes HCN as a metabolic product at concentrations of up to 0.3 mM [81]. Under these conditions, the heme-copper oxidases are inhibited. CIO likely has a role in allowing aerobic respiration under cyanogenic and microaerobic growth conditions [71,73,82]. Cyanide can be found in tissues infected with P. aeruginosa [83] that is consistent with the conclusion that CIO is required for full pathogenicity of P. aeruginosa in the cyanide-mediated paralytic killing of nematodes [84]. Mutation or overexpression of the cioAB genes of P. aeruginosa leads to temperature sensitivity for growth, difficulty exiting stationary phase, abnormal cell division and multiple antibiotic sensitivity [85].

There is no distinguishing feature in the sequences of the genes that allows one to differentiate CIO from other cytochrome bd family members. It is not yet clear whether the “short Q-loop” is a requirement for having the CIO phenotype or under what conditions such enzymes may or may not contain a stoichiometric content of heme d.

2. Physiological functions

The bioenergetic function of cytochrome bd is to conserve energy in the form of Δμ_H⁺ [41,45–50], although the H⁺/e⁻ ratio is 1, half the value of the A-subfamily heme-copper oxygen reductases such as the mitochondrial cytochrome c oxidase or cytochrome bo₃ from E. coli because the bd-type oxygen reductases do not pump protons [45,49,50].

Apart from PMF generation, cytochrome bd endows bacteria with a number of vitally important physiological functions. Cytochrome bd facilitates both pathogenic and commensal bacteria to colonize O₂-poor environments [86–89], serves as an O₂ scavenger to inhibit degradation of O₂-sensitive enzymes such as nitrogenase [90–98], and support anaerobic photosynthetic growth [99]. It is of interest to note that bd-type oxygen reductases predominate in the respiratory chains of bacteria that cause such diseases as bacillary dysentery [100], brucellosis [88,101], tuberculosis [87], pneumonia, life-threatening sepsis, meningitis [102], as well as Salmonella [103,104], Bacteroides [86], and Listeria monocytogenes [105] infections. There is a positive correlation between virulence of bacterial pathogens responsible for these diseases and level of cytochrome bd expression. Cytochrome bd enhances bacterial tolerance to nitrosative stress [106–111], contributes to mechanisms of detoxification of hydrogen peroxide in E.coli [112–114], suppresses extracellular superoxide production in Enterococcus faecalis [115], and is involved in the degradation of aromatic compounds in Geobacter metallireducens [116]. The A. vinelandii cytochrome bd might be directly involved in energizing Fe-siderophore transport or in reduction of Fe(III)-chelates and, thus, metal liberation in the cytoplasm [117]. As a source of oxidizing power, cytochrome bd-I in E. coli can support disulfide bond formation upon protein folding catalyzed by the DsbA-DsbB system [118], as well as the penultimate step of heme biosynthesis, the conversion of protoporphyrinogen IX into protoporphyrin IX, catalyzed by protoporphyrinogen IX oxidase [119].

The expression and membrane content of cytochrome bd in E. coli increase not only at low O₂ concentrations [120–122], but also under other stressful conditions, such as alkalization of the medium [123], high temperature [124,125], the presence of poisons in the environment (for instance, cyanide [126,127]), uncouplers-protonophores [123,128,129] and high hydrostatic pressure [130,131]. E. coli mutants defective in cytochrome bd are sensitive to H₂O₂ [125], zinc [127,132] and a self-produced extracellular factor that inhibits bacterial growth [133,134]. E. coli mutants that cannot synthesize cytochrome bd are also unable to exit from the stationary phase and resume aerobic growth at 37 °C [135,136].

Since cytochrome bd is found only in prokaryotes, including a number of human pathogens, the enzyme may be of interest as a drug target. A search for specific inhibitors of the bd-type oxygen reductases, which could be used in clinical practice, has been started [137,138]. An alternative, “positive” potential use of cytochrome bd might be for a therapy of respiratory chain deficiencies. It is known that mutations in genes encoding structural subunits of cytochrome bc₁ complex and cytochrome c oxidase can lead to severe neuromuscular and non-neuromuscular human diseases [139,140]. At the same time, it was reported that mixing purified cytochrome bd-I from E. coli with myxothiazol-inhibited bovine heart submitochondrial particles restores up to half of the original NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs [141]. Respiration bypassing the bc₁ complex is saturated at amounts of added bd-oxidase similar to that of other natural respiratory components in submitochondrial particles. Bacterial cytochrome bd-I tightly binds to the mitochondrial membrane and functions as an intrinsic component of the chimeric respiratory chain [141]. Thus, cytochrome bd, as well as AOX [142–144], might compensate for respiratory chain deficiencies in human cells.

3. Inhibitors

Table 1 shows the effect of different inhibitors on the respiratory activity of cytochrome bd from some bacteria. Quinol oxidase inhibitors can be divided into two groups: Q-like compounds acting at the Q binding site and heme ligands (e.g., cyanide, azide or NO) acting at the O₂ binding/reducing site. A specific feature of cytochrome bd is that it is much less sensitive to cyanide and azide than a heme-copper oxygen reductase like cytochrome bo₃ [27]. The lower sensitivity of cytochrome bd to anionic heme ligands may be a result of an elevated electron density on the central ion of iron due to breaking the conjugate π-electron structure in the d-type porphyrin ring and/or may point to a more hydrophobic environment of the O₂-reducing site. It was reported that cytochrome bd-I in E. coli is a bacterial membrane target for a cationic cyclic decapeptide gramicidin S (IC₅₀ ~5.3 μM, Table 1), although it has been generally accepted that the main target of gramicidin S is the membrane lipid bilayer rather than the protein components [145]. This finding can provide a new insight into the molecular design and development of novel gramicidin S-based antibiotics. The effect of gramicidin S on cytochrome bd-I and some other membrane-bound proteins could be the alteration of the protein structure through binding to its hydrophobic protein surface [145].

Table 1.

Effect of inhibitors on respiratory activity of cytochrome bd

Inhibitor	Bacterium
	E. coli(a)	B. stearo-thermophilus	A. vinelandii	Photobacterium phosphoreum
KCN or NaCN	2 mM (b) [27]	0.5 mM (e) [68]	-	62 μM (b) [288]
NaN3	400 mM (b) [27]	8.2 mM (e) [68]	-	40 mM (b) [288]
H2O2	120 mM (b) [27]	-	-	-
2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO)	7 μM (b) [27]	-	5–20 μM (d) [249]	8.2 μM (b) [288]
ZnSO4 or ZnCl2	60 μM (b) [27]	200 μM (e) [68]	-	2.7 μM (b) [288]
Piericidin A	15 μM (b) [27]	-	-	-
Antimicin A	50 μM, 80% (c) [285]	-	11 μM (d) [279,286]	-
Undecylhydroxydioxobenzothiazole (UHDBT)	20 μM, 18% (c) [285]	-	20 μM (d) [279,286]	-
(1,5-Dimethylhexyl)quinazolinamide	100 μM, 88% (c) [285]	-	-	-
(1-Methyldecyl)quinazolinamide	100 μM, 85% (c) [285]	-	-	-
Stigmatellin	200 μM, 14% (c) [285]	-	-	-
Nigericin	100 μM, 44% (c) [285]	-	-	-
Dibromothymoquinone	100 μM, 38% (c) [285]	-	-	-
Aurachin A	700 μM, 27% (c) [285]	-	-	-
Aurachin C	214 nM, 90% (c) [285]	-	-	-
Aurachin D	400 nM, 93% (c) [285]	-	-	-
decyl-aurachin D	-	-	13 nM (d) [249]	-
p-benzoquinone	-	120 μM (e) [68]	-	-
2,6-Dimethyl-p-benzoquinone	-	65 μM (e) [68]	-	-
Nitric oxide (NO)	100 nM (d) [106]	-	100 nM (d) [106]	-
Carbon monoxide (CO)	-	-	0.5–1 mM, 80% (g) [287]	-
Pentachlorophenol (PCP)	200 μM (d) [32]	-	-	-
2-Thenoyl trifluoroacetone (TTFA)	1 mM, 35% (f) [26]	-	-	-
Gramicidin S	5.3 μM (b) [145]	-	-	-

^(a)Data are referred to cytochrome bd-I.

^(b)IC₅₀ for ubiquinol-1 oxidase activity of the purified enzyme.

^(c)Concentration and % inhibition of duroquinol oxidase activity of cytochrome bd-containing membranes.

^(d)Inhibition constant (K_i) for ubiquinol-1 oxidase activity of the purified enzyme.

^(e)IC₅₀ for duroquinol oxidase activity of the purified enzyme.

^(f)Concentration and % inhibition of ubiquinol-1 oxidase activity of the purified enzyme.

^(g)Concentration and % inhibition of ascorbate-2,6-dichlorophenolindophenol oxidase activity of cytochrome bd-containing particles.

4. Genetics

4.1. Genes in E. coli encoding the protein subunits and assembly factors

Of the bd family, the best studied oxidase is cytochrome bd-I from E. coli. The two subunits of cytochrome bd-I are encoded by the cydAB operon [28,146,147] located at 16.6 min on the E. coli genetic map [146,148]. It was cloned [149] and sequenced [28]. The molecular weights of subunit I (CydA) – 57 kDa, and subunit II (CydB) - 43 kDa, determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis [26], are consistent with those of 58 and 42.5 kDa based on DNA sequence [28]. The enzyme subunits carry three hemes: b₅₅₈, b₅₉₅, and d [34,150]. Heme b₅₅₈ is located on subunit I (CydA), whereas hemes b₅₉₅ and d are likely to be in the area of the subunit contact [151]. CydA can be expressed and purified without CydB using mutant strains defective in cydB [152]. The purified CydA retains heme b₅₅₈ but lacks hemes b₅₉₅ and d [152]. In addition to the cydAB operon, the two other genes, cydC and cydD of the cydCD operon located at 19 min on the E. coli genetic map [132,153,154], are essential for the assembly of cytochrome bd-I [153–156]. CydC and CydD however are not subunits of cytochrome bd-I. It was shown that cydCD encodes a heterodimeric ATP-binding cassette-type transporter that is a glutathione transport system [157]. An orphan protein, YhcB, was proposed to be a third subunit of cytochrome bd-I [158], but this was later shown not to be the case [159].

In E. coli, a second cytochrome bd (bd-II) encoded by cyxAB genes (also named appBC or cbdAB) was identified [160]. The cyxAB genes, located at 22 min on the E. coli genetic map, are upstream from pH 2.5 acid phosphatase (appA) gene [160]. The cyxAB and appA genes constitute the complex operon. The cyxA and cyxB genes encode 58.1 kDa and 42.4 kDa integral membrane proteins, respectively. The deduced amino acid sequences of cyxA and cyxB genes reveal homologies of 60 and 57%, respectively, to subunit I (CydA) and subunit II (CydB) of cytochrome bd-I [160].

4.2. Regulation of gene expression in E. coli and other bacteria

Cytochrome bd-I is expressed by E. coli when the O₂ tension is low [120–122,161,162]. The expression of the cydAB operon is controlled by the two global transcriptional regulators, Arc and Fnr [121,161,163–169]. Arc is a two-component regulatory system that includes ArcA, a cytosolic response regulator, and ArcB, a transmembrane histidine kinase sensor. ArcA controls several hundred genes [170] and responds to the oxidation state of the Q pool which is sensed by ArcB [171]. ArcB is activated in response to the transition from aerobic to microaerobic growth and remains active during anaerobic growth. Upon stimulation, ArcB autophosphorylates and then transphosphorylates ArcA [171,172]. Under microaerobic conditions (i.e., O₂ tension of 2 to 15% of air saturation), the increased level of phosphorylated ArcA activates the cydAB operon [173]. Another global regulator, Fnr (an O₂-labile transcription factor regulating hundreds of genes), controls induction of anaerobic processes in E. coli [174,175]. The Fnr protein has a Fe-S cluster which serves as a redox sensor. The levels of the Fnr protein are similar under both aerobic and anaerobic conditions [165,176], but the protein is active only during anaerobic growth. The active Fnr protein represses cydAB operon during the transition to anaerobic conditions (i.e., O₂ tension of less than 2% of air saturation) [167,168,176].

Expression of cyxAB-appA operon (coding for cytochrome bd-II in E. coli) is induced by phosphate starvation and entry into a stationary phase [177]. The cyxAB genes can also be induced by anaerobic growth and this induction is controlled by transcriptional regulators AppY and ArcA but independent of Fnr, in contrast to cyd operon [177,178]. Cytochrome bd-II is likely to function under even more-O₂-limiting conditions than cytochrome bd-I [178]. Cytochrome bd-II has been partially purified [179], and contains two subunits by SDS-PAGE with apparent molecular weights 43 kDa (subunit I) and 27 kDa (subunit II). These subunits show no cross-reactivity to subunit-specific polyclonal antibodies directed against the subunits of cytochrome bd-I [179]. The spectral properties of cytochrome bd-II closely resemble those of cytochrome bd-I. Of the quinols tested as substrates, cytochrome bd-II utilizes menadiol as the preferred substrate (although ubiquinol-1, the most efficient in vitro substrate for cytochrome bd-I, was not tested). TMPD oxidase activity of cytochrome bd-II is much more sensitive to cyanide than that of cytochrome bd-I [179]. It was reported that though the electron flux through cytochrome bd-II can be significant, the enzyme does not contribute to the generation of the PMF [180]. Shepherd et al. [181] proposed that under conditions of an apparently fully uncoupled mode, E. coli can create PMF by means of consumption of intracellular protons in synthesis of γ-aminobutyric acid (GABA) and the generation of a pH gradient via uptake of glutamate and export of GABA by glutamate/GABA antiport.

In A. vinelandii, regulation of cytochrome bd expression is achieved by CydR (an Fnr homologue), which represses transcription of the cydAB genes [182]. The cydABCD operon coding for cytochrome bd in B. subtilis was reported to be activated by ResD and repressed by YdiH (Rex) and CcpA regulators [183–185]. Rex is also a repressor for the cydABCD operon in Streptomyces coelicolor [127]. ResD may activate the cydA gene in L. monocytogenes [105]. In Rhodobacter capsulatus, expression of cytochrome bd is likely controlled by RegA regulator [186].

5. Distribution and Evolution

The bd-family of oxygen reductases has a wide phylogenetic distribution with homologs found in at least one sequenced member of 18 bacterial phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Chlamydiae, Caldithrix, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Firmicutes, Nitrospirae, Planctomycetes, Proteobacteria, Thermi, Thermodesulfobacteria and Verrucomicrobia. To date no bd-family homologues have been detected in the following 12 bacterial phyla: Dictyoglomi, Elusimicrobia, Fibrobacteres, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Poribacteria, Synergistetes, Thermotogales, and candidate phyla NC10, TM7 and WWE1. A number of Archaea also encode bd-family homologues, with members of the family found in Crenarchaeota, Euryarchaeota [60] and Korarchaeota. Cytochrome bd-type oxygen reductases are very common is some phyla, such as the Proteobacteria and Actinobacteria, and sporadically distributed in others. Interesingly, bd-family homologues have been detected in many species described as strict anaerobes such as Methanosarcina barkeri, Methanosarcina acetivorans [60], Bacteroides fragilis [86], Desulfovibrio gigas [187–189], Desulfovibrio vulgaris Hildenborough [190], Geobacter metallireducens [116], Moorella thermoacetica [191] and Chlorobaculum tepidum [192].

Early work suggested that the bd-family of oxygen reductases is an ancient innovation, already present in the ancestor of both Bacteria and Archaea [193]. However it was recently reported that the family may have originated in Bacteria and was later acquired by Archaea via horizontal gene transfer [60,194]. Phylogenetic analysis of the bd-family showed that horizontal gene transfer plays a significant role in the distribution of the family, with many phyla acquiring cytochrome bd genes multiple times independently (Fig. 3).

Fig. 3.

The bd-family of oxygen reductases. An unrooted phylogenetic tree showing the relationships between 815 sequences of cytochrome bd oxidases. Members with the Q-loop insertion (long Q-loop) are shown in red. All other members of the family have the “short Q-loop”. A number of members from the purple clade have been classified as cyanide insensitive oxidases (CIO) with a low content of heme d. Cytochromes bd from Archaea are shown in blue and form two related clades. In contrast, cytochrome bd-type oxygen reductases from the Firmicutes (yellow) and Bacteroidetes (green) are highlighted to demonstrate the sporadic distribution of enzymes within these phyla which resulted from horizontal gene transfer.

Sequence analysis has demonstrated that subunits I and II have different rates of evolution, with subunit II evolving 1.2 times faster than subunit I [194]. The biological relevance of this asymmetrical evolution is currently unknown.

6. Membrane localization

Cytochrome bd is embedded in the prokaryotic cytoplasmic (plasma) membrane. It was reported that in E. coli, cytochrome bd-I is not evenly distributed within the plasma membrane, being concentrated in mobile (on the subsecond time scale) patches, of the order of 100 nm in diameter [195,196]. These clusters contain variable numbers of cytochrome bd-I tetramers [196]. Cytochrome bd in cyanobacteria [197–203] has been reported to also be located in the thylakoid membrane [200,201,203–207], though this has been disputed [208–211]. The presence of a bd-type PQH₂ oxidase in cyanobacterial thylakoid and/or cytoplasmic membranes may depend on culturing conditions and the light regime [201,206].

7. Cofactors and Substrates

7.1. Quinones

The nature of the quinols used by cytochrome bd as an electron donor is species-specific. For instance, in A. vinelandii and E. coli the cytochrome bd enzyme can oxidize ubiquinol (UQH₂), in B. stearothermophilus, the substrate is menaquinol (MQH₂). In E. coli, cytochrome bd-I can also oxidize MQH₂ [212,213], which replaces UQH₂ upon change of growth conditions from aerobic to anaerobic [166]. There is evidence that in cyanobacteria cytochrome bd is active as a plastoquinol (PQH₂) oxidase [200,201,203–206], although some reports have questioned this conclusion [208–210]. The presence or absence of bound Q in solubilized cytochrome bd-I from E. coli depends on the purification protocol. In some preparations of the purified enzyme, there is no apparently bound quinone [26,27,46,214] whereas others clearly contain bound quinone [41,215]. A stable semiquinone radical has been observed in the E. coli cytochrome bd-I [216,217].

7.2. Hemes

The two subunits of E. coli cytochrome bd-I carry three metal-containing redox-centers, two protoheme IX groups (hemes b₅₅₈ and b₅₉₅) and a chlorin molecule (heme d) which are in 1:1:1 stoichiometry per the enzyme complex. The enzyme contains no Fe-S cluster and no copper ion [218–222]. Heme b₅₅₈ is clearly located within subunit I. Both subunits are required for the assembly of heme b₅₉₅ and heme d, suggesting that these two hemes may reside at the subunit interface [151]. Heme b₅₉₅ appears to be oriented with its heme plane at ~55° to the plane of the membrane [223]. The millimolar extinction coefficients used commonly for the determination of the cytochrome bd concentration in E. coli and A. vinelandii are listed in Table 2.

Table 2.

Extinction coefficients used for determination of cytochrome bd concentration in E. coli and A. vinelandii.

Absorption spectrum	Heme	Wavelength pair (nm)	Δε (mM−1·cm−1)	Reference
E. coli (cytochrome bd-I)
Difference:	d	628–607	10.8	[37]
Reduced minus ‘as prepared’	d	628–651 a	27.9	[36]
	d	628–649 a	18.8	[27]
	b558	561–580	21	[36]
	b595	595–606.5	1.9	[36]
	all	429–700 b	303	[36]
CO/reduced minus reduced	d	642–622	12.6	[27]
	d	643–623	13.2	[48]
Absolute:
Reduced	d	628–670	25	[41]
‘As prepared’	all	414–700 b	223	[36]
A. vinelandii
Difference:
Reduced minus ‘as prepared’	d	628–605	9.5	[241]
	d	629–608	12	[257]
	d	629–650 a	27	[257]
CO/reduced minus reduced	d	622–642	18	[257]

^a,bThese values cannot be recommended for determination of cytochrome bd concentration since

^athe ‘as prepared’ enzyme contains varying amounts of the ferrous heme d-oxy complex that absorbs at 649–651 nm, and

^bthe intensity of the Soret band is variable depending on the purity of the preparation.

7.2.1. Heme b₅₅₈

Heme b₅₅₈ has been shown to be located within subunit I by expressing subunit I (cydA) in the absence of subunit II (cydB) and showing that the isolated subunit I contains heme b₅₅₈ [152]. Antibodies directed against subunit I [61,63], as well as selective proteolysis of this subunit [62,64], inhibit UQH₂ oxidase activity of cytochrome bd-I. These findings suggest that heme b₅₅₈ is associated with subunit I and is involved in QH₂ oxidation. The α- and β-bands of the reduced heme b₅₅₈ at room temperature reveal maxima at 560–562 and 531–532 nm, respectively (Table 3) [150,224,225]. The maximum and minimum of the γ-band in the “reduced minus oxidized” difference absorption spectrum are 429.5 and 413 nm, respectively (Table 3) [225]. Heme b₅₅₈ is low-spin hexacoordinate [37], and amino acid residues His186^** and Met393 of subunit I (E. coli cytochrome bd-I) have been identified as its axial ligands [226–228]. The location of heme b₅₅₈ is predicted to be near the periplasmic surface [67,229].

Table 3.

Spectral properties of cytochrome bd-I from E. coli. Shown are wavelengths (nm) and extinction coefficients (in parentheses, mM⁻¹·cm⁻¹) for “reduced-minus-oxidized” difference absorption spectra. Data are taken from reference [225].

	Heme b558	Heme b595	Heme d
Maxima	429.5 (90), 531.5 (5.8), 561 (17.2)	439 (113), 561.5 (8.2), 594 (5.3)	430 (30), 629 (18)
Minima	413 (−40), 497 (−4.3), 545 (~0)	400 (−37), 500 (−3.6), 643 (−1.18)	405 (−23), 468 (−6.3), 657.5 (−2.7), 739±2 (−2.4)
Isosbestic points	421, 450, 518, 573	422, 457, 535, 613	418.5, 449, 602, 648

7.2.2. Heme b₅₉₅

The spectrum of heme b₅₉₅ is similar to that of catalases and peroxidases containing pentacoordinate (high-spin) protoheme IX [150]. Heme b₅₉₅ has an α-band at 594–595 nm and β-band at 560–562 nm in the difference absorption spectrum (Table 3) [150,224,225]. A trough at 643–645 nm in the difference spectrum of heme b₅₉₅ is indicative of the disappearance in the reduced heme b₅₉₅ of an absorption feature due to charge transfer from the Fe to the ligand, characteristic of oxidized high-spin heme b, as in the case of peroxidases. The γ-band of ferrous heme b₅₉₅ is characterized by a maximum at ~440 nm as clearly revealed by femtosecond spectroscopy [38]. The maximum and minimum of the γ-band in the difference “reduced minus oxidized” absorption spectrum are 439 and 400 nm, respectively (Table 3) [225]. Heme b₅₉₅ is high-spin pentacoordinate [37], ligated by His19 of subunit I [230] and located near the periplasmic surface [67,229]. The role of heme b₅₉₅ remains obscure. It is proposed that heme b₅₉₅ participates in the reduction of O₂ forming, together with heme d, a di-heme O₂-reducing site, somewhat similar to the heme/Cu O₂-reducing site in heme-copper oxidases [35–41,43,231]. In favor of this hypothesis is the finding that the CD spectrum of the reduced wild type cytochrome bd in the Soret band shows strong excitonic interaction between ferrous hemes d and b₅₉₅ [42]. Modeling the excitonic interactions in the absorption and CD spectra yields an estimate of the Fe-to-Fe distance between heme d and heme b₅₉₅ to be about 10 Å [42]. In the opinion of some, the function of heme b₅₉₅ is limited to transferring an electron from heme b₅₅₈ to heme d [232,233], whereas others have postulated that heme b₅₉₅ can form a second site capable of reacting with O₂ [218,234].

7.2.3. Heme d

Heme d is a chlorin-type molecule [235]. The α-band of the reduced heme d in the absolute absorption spectrum of E. coli cytochrome bd-I shows a peak at 628–630 nm. However, upon isolation of the enzyme, heme d is in the stable oxygenated (O₂-ligated ferrous) form, which is characterized by an absorption band with a maximum at 647–650 nm in the absolute absorption spectrum [236–239]. The affinity of ferrous heme d for O₂ is indeed high, showing the K_d(O2) values of 0.28 μM and 0.5 μM for the enzymes from E. coli and A. vinelandii, respectively [240,241]. The maximum and minimum of the γ-band in the difference “reduced minus oxidized” absorption spectrum are 430 and 405 nm, respectively (Table 3) [225].

Remarkably, the spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b [225]. Heme d is predicted to be located near the periplasmic surface [67,229], and is the site for capturing and, subsequently, reducing O₂ to H₂O. In the absence of external ligands, heme d is in the high-spin state with an open coordination site for binding O₂. The nature of the axial ligation of heme d to the protein, or even whether there is an axial ligand provided by the protein, is unclear. It has been claimed that the reduction of cytochrome bd is associated with binding of an endogenous protein ligand to heme d [242]. The oxidized heme d may or may not be ligated to an endogenous protein substituent. Resonance Raman and ENDOR studies indicate that the ligand is not histidine, cysteine or tyrosinate, but that the single axial ligand is either a weakly coordinating protein donor or a water molecule [230,243,244]. In contrast, EPR studies indicated that the heme d axial ligand is histidine in an anomalous condition or some other nitrogenous amino acid residue [245]. Finally, it has been suggested that Glu99 of subunit I is a prime candidate for such a role [214,246].

7.3. Heme redox potentials

The apparent values for the midpoint redox potentials of hemes b₅₅₈, b₅₉₅ and d for the bd enzymes solubilized in n-dodecyl-β-D-maltoside at pH 7.0 (E_m) are respectively +176, +168, +258 mV (E. coli bd-I) and +166, +251, +310 mV (A. vinelandii) [241]. These are within the range of the values reported earlier for E. coli [219,220,224,247,248] and A. vinelandii [249]. Notably, the E_m value of heme b₅₅₈ can depend on the detergent used for solubilization [248]. In particular, octylglucoside and cholate cause a large decrease in the E_m value of heme b₅₅₈, and this correlates with the reversible inactivation of the enzyme [248]. The E_m values of all three heme components of cytochrome bd are sensitive to pH between pH 5.8 and 8.3 with a E_m/pH of −61 mV for heme d and −40 mV for hemes b₅₅₈ and b₅₉₅, indicating that reduction of cytochrome bd is accompanied by enzyme protonation [248]. A recent study [225] revealed a significant redox interaction between heme b₅₅₈ and heme b₅₉₅, whereas the interaction between heme d and either both hemes b appears to be rather weak. However, the presence of heme d itself decreases the much larger interaction between the two hemes b [225].

8. Proposed structure

The X-ray structure of cytochrome bd has not been determined. Conventional studies of the protein topology in the membrane suggest that all three hemes are located near the periplasmic side of the membrane [67,229], although an alternative view also exists [250,251]. Fig. 4 shows topological models of subunits I (CydA) and II (CydB) of cytochrome bd-I from E. coli [213]. Both subunits are integral membrane proteins. Subunit I consists of nine transmembrane helices with the N-terminus in the periplasm and the C-terminus in the cytoplasm [67]. Subunit II is composed of eight transmembrane helices with both N- and C-termini in the cytoplasm [67]. The Q-loop in subunit I connects transmembrane helices 6 and 7, and is directly involved in QH₂ binding and oxidation [61–66]. Thus the QH₂-oxidizing site in cytochrome bd is located on the periplasmic side of the membrane. Cytochrome bd-I from E. coli is proposed to contain a single site for the binding and oxidation of quinol [65,66,252]. However, evidence for a second quinone binding site in cytochrome bd from Corynebacterium glutamicum has also reported [69].

Fig. 4.

Proposed topology of subunits I and II of cytochrome bd-I from E. coli. The axial ligands of heme b₅₉₅ (H19) and heme b₅₅₈ (H186 and M393) in subunit I are highlighted. The model is based on the data reported in [67,213,229].

Using a set of 815 sequences of genes encoding cytochrome bd, a number of residues in subunit I are totally (>99%) conserved [213]. These residues include those which are identified as ligands to the heme components of the enzyme. In addition, since the active site of O₂ reduction is located near the periplasmic surface and protons for H₂O production are taken from the bacterial cytoplasm, there must be at least one transmembrane proton-conducting pathway to convey protons from the cytoplasm to the heme b₅₉₅/heme d site [41,46,48,67] (Fig. 5). Several polar or ionizable residues that are highly conserved in the bd-family have been postulated to be a part of this putative proton channel.

Fig. 5.

Scheme for electron and proton transfer pathways in cytochrome bd-I from E. coli. There are two protonatable groups, X_P and X_N redox-coupled to the heme b₅₉₅/heme d active site. A highly conserved E445 was proposed to be either the X_P group or the gateway in a channel that connects X_P with the cytoplasm or the periplasm [41]. A strictly conserved E107 is a part of the channel mediating proton transfer to X_N from the cytoplasm [48].

The residues that are totally conserved within the entire bd-family include His19 (the heme b₅₉₅ axial ligand [230]), His186 and Met393 (the heme b₅₅₈ axial ligands [226–228]), Lys252 and Glu257 (involved in QH₂ binding [66]), Arg448 (unknown function), and Glu99, Glu107, and Ser140 (proposed to be components of a proton channel [48,67] and important for heme binding in the heme d/heme b₅₉₅ di-heme site [213,214]). Slightly less conserved (95–99%) are Glu445 (required for charge compensation of the b₅₉₅/d O₂-reducing site upon its full reduction by two electrons [41]), Asn148 (plausible component of a proton channel), and Arg9 (unknown function) [213]. Somewhat less conserved (~ 85%) are Arg391 (stabilizes the reduced form of heme b₅₅₈ [253]) and Asp239 (unknown function), however these residues are totally conserved within the A subfamily of cytochromes bd [213]. Other conserved residues are glycines, prolines, phenylalanines, or tryptophans, which may play structural roles. There is only one totally (>99%) conserved residue (Trp57) in subunit II [213]. Within the subfamily of bd-type oxygen reductases which have the “long Q-loop”, Arg100, Asp29, and Asp120 of subunit II are totally conserved and Asp58 (subunit II of E. coli cytochrome bd-I) is either an aspartate or glutamate [213]. The N-terminal portion of subunit II has been suggested to be involved in the binding of heme d/heme b₅₉₅ [213,254].

Fig. 3 shows an unrooted tree showing the relative sequence relationships of 815 sequences of cytochrome bd from the genomes of Bacteria and Archaea. It is seen in Fig. 3 that the “long Q-loop” members form a phylogenetic clade distinct from the other members of the family. This is most likely due to an insertional event within the Q-loop. This subfamily contains many, but not all, of the cytochrome bd oxygen reductases from Proteobacteria (including E. coli). Also shown in Fig. 3 are two clades that define the bd-family members found in Archaea. In contrast, the bd-family oxygen reductases found in Firmicutes or Bacteroides are distributed widely among the phylogenetic groups shown in Fig. 3. This illustrates the large role played by horizontal gene transfer in the distribution of the bd-type oxygen reductases.

9. Binding of ligands (other than O₂)

Since hemes d and b₅₉₅ in cytochrome bd are in the high-spin pentacoordinate state, they could potentially bind ligands. One may anticipate that the enzyme in the reduced state binds electroneutral molecules like O₂, CO, and NO, whereas the oxidized cytochrome bd prefers ligands in the anionic form such as cyanide and azide. Heme d binds ligands readily whereas the ligand reactivity of heme b₅₉₅ is minor despite the fact that this is a high spin heme [37,39,255]. Heme b₅₅₈, although a low-spin hexacoordinate, may also bind ligands to some extent (e.g., CO or cyanide) [37,255]. Such a marginal reactivity is possibly due to weakening the bond of the methionine axial ligand (Met393) to heme b₅₅₈ iron caused by the isolation procedure and/or protein denaturation [255].

9.1. Carbon monoxide

Addition of CO to the three-electron reduced form of cytochrome bd, denoted as R³, causes a red shift of the 628 nm heme d band and the increased absorption around 540 nm in the visible, as well as a distinctive W-shaped difference spectrum in the Soret region [37,39,150,255–257]. The W-shaped feature is due to a small bandshift of unligated heme b₅₉₅ induced by CO interaction with the nearby heme d [38,40,43]. Only a small fraction of heme b₅₉₅ (<5%) in cytochrome bd binds CO at room or low temperature [37,39]. The apparent K_d for the CO-heme d complex with the fully reduced (R³) cytochrome bd-I from E. coli was determined to be ~80 nM [255]. The R³ cytochrome bd can form a photosensitive heme d-CO complex [258]. Flash photolysis of CO bound to heme d at cryogenic temperatures results in a redistribution of CO such that as much 15% of heme b₅₉₅ is bound to CO, showing the proximity of these two hemes [35]. Following flash-photolysis of the heme d-CO complex in the fully reduced enzyme (R³) at room temperature, CO recombines with ferrous heme d proportionally to the external CO concentration with a second order bimolecular rate constant of 10⁸ M⁻¹ s⁻¹ (Table 4) [43,222,249,259].

Table 4.

Kinetic and thermodynamic parameters for reaction of cytochrome bd with gaseous ligands at room temperature.

	E. coli (cytochrome bd-I)						A. vinelandii
	O2		CO		NO		O2	CO
	R1-O2	R3-O2	R1-CO	R3-CO	R1-NO	R3-NO	R3-O2	R1-CO	R3-CO
kon (M−1 s−1)		2 × 109 b,e		8 × 107 b,f			2 × 109 h 2.8 × 108 i	1 × 108 g	1.5 × 108 g
koff (s−1)	78a		4.2a	6a	0.036a	0.133a
Kd (nM)	280c			80d

^a[107];

^b[222];

^c[240];

^d[255];

^e[47];

^f[43];

^g[249];

^h[241];

ⁱ[278].

The one-electron reduced form of the enzyme (R¹) can also be examined. Since heme d has a substantially higher midpoint potential than the other two heme components, heme d is the only heme reduced in the R¹, or mixed-valence, state of the ‘as prepared’ enzyme. Upon reaction with CO, one gets the CO-heme d adduct (b₅₅₈³⁺b₅₉₅³⁺d²⁺-CO) [38,40,43,48,107,249]. After flash photolysis of the R¹-CO complex, a substantial fraction of the CO flashed off heme d²⁺ gets trapped inside the protein and undergoes geminate recombination with heme d²⁺ on the pico- and nanosecond time scale [38,43]. The data indicate that the redox state of heme b₅₉₅ controls the pathway for ligand (CO) transfer between heme d and the bulk phase, which is open when heme b₅₉₅ is reduced but closed when heme b₅₉₅ is oxidized [38,43,107].

9.2. Nitric oxide and other nitrogen-containing ligands

A number of small nitrogen-containing molecules can react with R³ cytochrome bd from E. coli and A. vinelandii. NO₃⁻, NO₂⁻, N₂O₃²⁻ (trioxodinitrate), NH₂OH and NO, when added to membranes containing cytochrome bd or the purified enzyme, give rise to decrease in amplitude and shift of the 630 nm peak of ferrous heme d to 641–645 nm [31,37,106,107,218,245,257,260–264]. It appears that all of these ligands result in chemical reactions, forming the same or a very similar heme-nitrosyl compound [31], e.g., heme d^{2 +}-NO adduct. It has also been suggested that a heme b₅₉₅²⁺-NO adduct can be observed upon adding nitrite to cytochorme bd in membranes [218].

Cytochrome bd can also produce a stable complex with NO in the R¹ state, in which ligand bound heme d is reduced while the b hemes are oxidized [107,245]. The rates of NO dissociation from heme d²⁺ in both R³ and R¹ states of cytochrome bd were determined [107]. In the R³ state, NO dissociates from heme d²⁺ at an unusually high rate, k_off = 0.133 s⁻¹ [107], which is ~30-fold higher than the off-rate measured for the ferrous heme a₃ of the mitochondrial cytochrome c oxidase (k_off = 0.004 s⁻¹ [265]). These data are consistent with the proposal that, in the heme–copper oxidases, Cu_B acts as a gate controlling ligand binding to the heme in the active site [266]. Another remarkable feature of NO dissociation from cytochrome bd is that the k_off value in the R¹ state (0.036 s⁻¹), although still quite high, is significantly lower than that measured with the R³ enzyme [107] (Table 4). These data show that the redox state of heme b₅₉₅ controls the kinetic barrier for ligand dissociation from the active site of cytochrome bd, similar to the observations with CO dissociation from ferrous heme d [38,43,107]. The unusually high NO dissociation rate from cytochrome bd may explain the observation [106] that the NO-poisoned cytochrome bd recovers respiratory function much more rapidly than a heme–copper oxygen reductase. It is postulated that expression of bd-type, instead of heme–copper-type oxygen reductase, enhances bacterial tolerance to nitrosative stress, thus promoting colonization of host intestine or other microaerobic environments [107,108]. It was reported that, apart from ferrous heme d, NO can also react with the oxoferryl and ferric state of heme d, yielding the oxidized nitrite-bound heme d and the nitrosyl adduct, respectively [110,111].

9.3. Cyanide

Reaction of ‘air-oxidized” cytochrome bd with KCN causes the decay of the ferrous heme d oxy-complex [267–273]. Cyanide-induced changes to the EPR-spectrum include a low-spin signal and, after prolonged incubation, a second weak low-spin signal that may indicate some interaction of cyanide with heme b₅₉₅ [220,257,274]. A simple and fast method for conversion of the oxygenated enzyme into the O form with the use of lipophilic electron acceptors [239] allowed us to study the interaction of cyanide with the homogenous oxidized preparation of cytochrome bd [37]. The MCD spectrum of the O cytochrome bd-I from E. coli is dominated by an asymmetric signal in the Soret. Submillimolar cyanide has no effect on the initial MCD spectrum. 50 mM KCN induces minor changes of the MCD signal in the Soret band, which can be modeled as transition of a part of the low-spin heme b₅₅₈ (15–20%) to its low-spin cyano-complex [37]. There is no evidence of the interaction of high-spin ferric heme b₅₉₅ with the ligand [37]. On the contrary, based on the EPR spectra, Tsubaki et al. [36] proposed that the treatment of ‘air-oxidized’ cytochrome bd with cyanide results in a cyanide-bridging species with a “heme d³⁺–C=N–heme b₅₉₅³⁺” structure. However the authors [36] did not account for the electron released from heme d upon cyanide binding to ‘as prepared’ cytochrome bd. Resonance Raman studies suggest that heme d is in the high-spin pentacoordinate state when it is compounded with cyanide [230,275]. This would require either that the endogenous axial ligand to heme d is displaced by cyanide, maintaining a high-spin pentacoordinate state, or that there is no endogenous axial ligand to heme d in the fully oxidized form of the enzyme.

9.4. Hydrogen peroxide

Addition of excess H₂O₂ to E. coli membranes containing cytochrome bd-I [276] and the purified enzyme in the ‘as prepared’ [231,237] or the O [46,231,277] states gives rise to an absorption band at ~680 nm. The reaction of H₂O₂ with the O cytochrome bd also induces a red shift of the γ-band [231,277]. H₂O₂ binds to ferric heme d with an apparent K_d value of 30 μM, but it seems not to interact with heme b₅₉₅ [231,277]. The O cytochrome bd reacts with H₂O₂ with a second order rate constant of 600 M⁻¹ s⁻¹. The decay of the H₂O₂-induced spectral changes upon addition of catalase (k ~ 10⁻³ s⁻¹) is about 20-fold slower than expected for dissociation of H₂O₂ from the complex with heme d assuming a simple reversible binding of peroxide [277]. This suggests that the interaction of H₂O₂ with cytochrome bd is essentially irreversible, giving rise to the F state of heme d [277]. The assignment of the compound 680 to the F state of heme d is confirmed by resonance Raman spectroscopy data [221]. Heme d in the F state is suggested to be high-spin pentacoordinate [275].

10. Proposed catalytic mechanism

As discussed above, under physiological conditions cytochrome bd from different prokaryotes likely oxidizes UQH₂, MQH₂ or PQH₂. In vitro a bd-type oxygen reductase can also utilize short chain ubiquinols, menadiol, duroquinol, and artificial electron donors such as TMPD. Of the in vitro substrates, ubiquinol-1 (plus excess dithiothreitol) shows the highest turnover numbers [248,278]. The activity of the purified oxidase depends on the nature of the detergent in which the enzyme is solubilized. Cytochrome bd-I from E. coli is inactive in octylglucoside or cholate but shows high activity in Tween-20, Triton X-100 [248] or N-lauroyl-sarcosine [106]. The ubiquinol-1 oxidase activity of cytochrome bd-I has a broad optimum above pH 7.5 but decreases at more acidic pH values [248]. Cytochrome bd possesses three distinct active sites - for QH₂ oxidation, TMPD oxidation and O₂ reduction. All the three sites seem to be located at or close to the periplasmic surface of the membrane. Electrons donated from QH₂ transfer to heme b₅₅₈ and then to the b₅₉₅/d di-heme site, whereas electrons donated from TMPD transfer directly to the b₅₉₅/d site bypassing the QH₂-binding site and heme b₅₅₈ [62,279].

10.1. Mechanism of generation of the proton motive force

Cytochrome bd from E. coli and A. vinelandii was reported to generate a transmembrane electric potential both in single turnover [41,46–48] and under multiple turnover [27,44,280] conditions (H⁺/e⁻ ~ 1 [34,45,49,50]; q/e⁻ ~ 1 [281]). When reconstituted into liposomes, cytochrome bd generates an uncoupler-sensitive transmembrane voltage difference with a value of 160–180 mV (negative inside) [27,44]. The QH₂ molecule generated by the dehydrogenases of the respiratory chain can diffuse laterally within the bilayer, finding its way into the QH₂ oxidizing site located near the outer side of the membrane. Upon oxidation of QH₂, two protons are released into the periplasmic space, and two electrons are transferred through heme b₅₅₈ to the b₅₉₅/d O₂-reducing site, also located near the periplasmic surface of the membrane. The four protons used for O₂ reduction are taken up from the cytoplasm. Single-turnover electrometric experiments show that the generation of the membrane potential is associated with electron transfer from heme b₅₅₈ to the b₅₉₅/d active site [41,46–48]. However, since all of the three hemes are likely located close to the periplasmic side of the membrane [67,229], the electron transfer itself is expected to be parallel to the membrane surface and, therefore, cannot be electrogenic [46]. Rather, it is proposed that electron transfer from heme b₅₅₈ to the b₅₉₅/d active site is coupled to vectorial proton transfer from the cytoplasm towards the active site on the opposite (periplasmic) side of the membrane [41,46–48]. The latter implies that there must be a proton-conducting channel connecting the cytoplasm to the b₅₉₅/d active site [41,46,48] (Fig. 5). The transmembrane potential originates primarily from protons moving from the cytoplasm to the O₂-reducing site on the opposite side of the membrane, and this accompanies electron transfer from heme b₅₅₈ to the b₅₉₅/d active site. As shown in Fig. 5, it is proposed that near the b₅₉₅/d active site there are two protonatable sites (X_P and X_N) that are accessible to the cytoplasm via a proton-conducting channel.

10.2. Reaction of the fully reduced enzyme (R³) with O₂

The reaction of the R³ cytochrome bd with O₂ has been studied using the flow-flash method [282] by means of spectroscopic and electrometric techniques [41,46–48,222]. Recording absorption spectra and membrane potential development with 1 μs time resolution resolves the sequence of the catalytic intermediates and establishes which catalytic steps are linked to electric potential generation [47]. The scheme for this reaction is presented in Fig. 6 (top panel). The initial complex of R³ cytochrome bd with CO (R³-CO) is photolyzed (the photolysis details are shown in Fig. 6, bottom panel) in the presence of O₂. The unliganded R³ enzyme, generated by the CO-photolysis, binds O₂ very rapidly, forming the ferrous heme d oxy species (A³). The R³→A³ transition is not electrogenic and its rate is proportional to [O₂] (k_on = 1.9 × 10⁹ M⁻¹·s⁻¹ [47,222]). The A³ formation is followed by electron transfer from heme b₅₉₅ to form state P. The A³→P transition occurs with τ = 4.5 μs and is also nonelectrogenic [47]. Thus, electron transfer from heme b₅₉₅ to heme d is not coupled to membrane potential generation [41,47]. It is proposed that P is a peroxy complex of ferric heme d [47]. If this is the case, the bound peroxide is likely not to be in the anionic form but at least singly protonated. The proton may come from one of two postulated protonatable groups, X_P and X_N, near the b₅₉₅/d di-heme active site upon oxidation of the hemes [41]. P is further converted into F upon electron transfer from heme b₅₅₈ with τ = 48 μs. Formation of F is coupled to generation of a membrane potential [41,46–48] due to the accompanying proton transfer through the proposed proton channel (Fig. 5). At the F stage, the b-type hemes are in a ferric state and heme d in an oxoferryl state. When cytochrome bd contains bound QH₂, the reaction proceeds further to form the O enzyme. The F→O transition occurs with τ = 1.1 ms and is electrogenic as well [41,47] since this also involves electron transfer from heme b₅₅₈ to the b₅₉₅/d active site with the accompanying proton transfer.

Fig. 6.

Top: Scheme for reaction of fully reduced cytochrome bd with O₂. The three rhombuses represent hemes b₅₅₈, b₅₉₅, and d, respectively. The minus sign denotes that the heme is in the ferrous state. Bottom: Photolysis of CO from heme d in the fully reduced enzyme. Two different configurations of dissociated CO in the enzyme (d……CO_i, i=I, II) are proposed [43]. The state (d + CO) denotes a state where CO escaped from the enzyme.

Cytochrome bd can bind O₂ being in the R¹ state. Remarkably, in this reaction, the dependence of the rate of O₂ binding on [O₂] is hyperbolic thus revealing a saturation behavior. This is not observed for O₂ binding to the R³ enzyme [241]. It is speculated that the R¹ enzyme exists in the two different conformations in equilibrium, but only one of these forms binds to O₂. When in the “closed” conformation, cytochrome bd provides no access for O₂ to heme d²⁺, whereas in the “open” conformation, O₂ binds easily. The R³ enzyme is always in the open conformation [241].

10.3. Catalytic cycle

Several relatively stable forms of cytochrome bd corresponding to the intermediates of the catalytic cycle have been identified. Under aerobic conditions, cytochrome bd is predominantly in the one-electron-reduced state bound to O₂ (A¹), with lesser amounts of the F and O forms. Under anaerobic conditions, the reduced forms of the enzyme lacking an O₂ ligand with one (R¹) and three (R³) electrons can be generated and examined. A short-lived complex of the three-electron reduced cytochrome bd with O₂ (A³) [46,47,222,241], an “peroxide” intermediate P [47] and an oxoferryl compound F [46,47,222] can be sequentially formed (Fig. 6). Turnover intermediates of E. coli cytochrome bd-I detected at steady-state are A¹ and F species (~40% each) and, to a lesser extent (~20%), a species with ferric heme d and possibly one electron on heme b₅₅₈ (R¹) [283]. These data differ from those obtained with mammalian cytochrome c oxidase, in which oxygenous intermediates were not found to be populated at detectable levels under similar conditions [284]. A plausible scheme of the catalytic cycle of cytochrome bd is shown in Fig. 7.

Fig. 7.

Cytochrome bd catalytic cycle. The scheme is based on the reports of Junemann et al. [278], Kavanagh et al. [289], Matsumoto et al. [252], Belevich et al. [47], Yang et al. [290], and Borisov et al. [283]. Solid arrows show the natural catalytic reaction pathway. Dotted arrows indicate transitions that are not being part of the catalytic cycle can be observed experimentally. The O form of the enzyme is most likely not to be an intermediate of the catalytic cycle [290]. Intermediates populated at steady-state [283] are highlighted in grey.

10.4. Role of heme b₅₉₅

Exogenous ligands added to cytochrome bd bind to heme d but do not bind to a majority of the heme b₅₉₅ population [31,37,39,255]. Heme b₅₉₅, although in the high-spin pentacoordinate state, is resistant to interaction with the classical ligands of high-spin iron-porphyrin complexes. It cannot be ruled out that despite the high-spin pentacoordinate state of the iron-porphyrin group, the specific features of the protein environment are such that this redox cofactor is protected from interaction with ligands. In such case, the participation of heme b₅₉₅ in O₂ reduction in cooperation with heme d is unlikely and its role would be limited to the transfer of an electron to heme d. A more likely explanation is the following: (1) both heme b₅₉₅ and heme d potentially can bind ligands; (2) the hemes are located close to each other forming a di-heme active site; (3) the spatial proximity of hemes b₅₉₅ and d results in steric restrictions allowing the di-heme site to bind only one ligand molecule; (4) heme d has a higher affinity for ligands than heme b₅₉₅, in which case the final result observed upon addition of a ligand will always be the ligand binding to heme d, whereas heme b₅₉₅ will remain mainly in the unliganded state [37,39,231,255]. The data on the redox coupling of the two hemes to the same ionizable groups [41], and the migration of CO within the protein from heme d to heme b₅₉₅ at cryogenic temperatures [35] are in agreement with this proposal. Modeling the excitonic interactions in absorption and CD spectra of cytochrome bd yields an estimate of the Fe_d-to-Fe_b595 distance of about 10 Å [42]. This is markedly larger than that for the Fe/Cu_B pair in heme-copper oxidases (4–5 Å). If this is the case, heme b₅₉₅ cannot be a functional analogue of Cu_B. A possible role of heme b₅₉₅, apart from electron delivery to heme d and/or to an oxygenated intermediate form of heme d, would be as a binding site for hydroxide produced from heme d-bound O₂ upon reductive cleavage of the O-O bond [42].

11. Conclusion

There are at least two reasons why cytochromes bd may be of interest. First, they are found in many pathogenic bacteria and there is growing evidence for a positive correlation between the virulence and the level of cytochrome bd expression. We hope that our knowledge on the structure and function of the bd enzymes will provide new tools to combat diseases caused by pathogens, for instance, by using a bacterial bd-type respiratory oxygen reductases as a drug target. Second, it would be useful to know what are the common features and the differences between the mechanisms of O₂ reduction to H₂O by cytochromes bd and heme-copper oxidases. Such a comparison could allow us to gain further insight into the elements essential for proton pumping coupled to the redox reaction inherent in heme-copper oxidases.

Research highlights.

• Physiological functions and genetics of cytochrome bd terminal oxidases reviewed.

• Structural and catalytic properties of cytochromes bd discussed.

• Phylogenetic analysis of cytochromes bd and their homologues presented.

Abbreviations

AOX

alternative oxidase

CIO

cyanide-insensitive quinol oxidase

E_m

apparent midpoint redox potential

IC₅₀

the half maximal inhibitory concentration

PMF

proton motive force

TMPD

N,N,N′,N′-tetramethyl-p-phenylendiamine

Q

quinone

QH₂

quinol

UQH₂

ubiquinol

MQH₂

menaquinol

PQH₂

plastoquinol

A¹

one electron-reduced O₂-bound species

A³

fully reduced O₂-bound species

R¹

one electron-reduced species

R³

fully reduced species

O

fully oxidized species

F

oxoferryl species

P

peroxide-bound species

Δμ_H⁺

transmembrane difference in the electrochemical H⁺ potentials

τ

time constant reciprocal of rate constant (t_1/e)