The cytochrome bd respiratory oxygen reductases

Abstract

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.

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.

Fig. 2

Proposed cytochrome bd model.

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.

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].

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].

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.

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.