Interaction of Cytochrome bd with Carbon Monoxide at Low and Room Temperatures

Abstract

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.Cytochrome bd is a terminal oxidase present in the respiratory chains of many bacteria (reviewed in Ref. 1, see an evolutionary tree in Ref. 2). The enzyme catalyzes reduction of molecular oxygen to water by ubiquinol or menaquinol as natural electron donors (3-5) but shows no sequence homology to the heme-copper quinol oxidases. Also, in contrast to these oxidases, cytochrome bd does not contain copper and, although generating ⌬ (6 -9), does not pump protons (10).Cytochrome bd-type oxidases purified from Azotobacter vinelandii or Escherichia coli consist of two subunits and carry three iron-porphyrin groups: low spin heme b 558 , high spin heme b 595 , and a chlorin-type high spin iron-porphyrin group (heme d). All three redox centers are proposed to be located near the periplasmic side of the membrane (2). Heme b 558 is directly involved in ubiquinol oxidation (11,12). Heme d binds O 2 and is involved in the trapping and reduction of oxygen (3). The specific role of heme b 595 is still a matter of debate. One obvious possible role is the transferrence of electrons from heme b 558 to heme d (13-15). However, because heme b 595 is high spin, it is tempting to consider its involvement in dioxygen reduction; it was proposed that hemes b 595 and d might form a binuclear dioxygen reduction center analogous to the hemecopper oxygen-reducing site in the cytochrome aa 3 -or bo 3 -type oxidases (16 -19). In such a case, one might expect heme b 595 to react with other exogenous ligands such as CO or NO as is typical of most high spin hemoproteins. Surprisingly, despite a long history of such studies, this essential and apparently simple question has not been answered by the apparently conflicting data. The intricate line shape ...

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

“…This is in line with the proposal of physical proximity of hemes d and b 595 and their functional cooperation in the O 2 -reducing site (31)(32)(33)(34)(35)(36)(37).…”

Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli

“…They are widely distributed in pathogenic bacteria and responsible for a number of vitally important physiological functions [29][30][31] which include conferring resistance to NO stress [32][33][34][35][36]. Earlier CO was used as a ligand for probing the cytochrome bd active site [12,[14][15][16]20,37]. The ligand was reported to react to heme d and at high concentrations to a fraction of heme b 558 [14,37].…”

“…Their reaction mechanism and interactions with small molecules like CO and NO have been intensively studied [1][2][3][4][5]. The tri-heme cytochrome bd oxidases, which refer to ironchlorine proteins [6][7][8], contain no copper [9][10][11] and are thought to bind, activate and reduce O 2 using a di-heme active site composed of the high-spin pentacoordinate hemes d and b 595 [12][13][14][15][16][17][18][19][20][21]. The third heme, low-spin hexacoordinate b 558 , transfers electrons from the natural electron donor quinol towards the di-heme site [22].…”

Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site

“…However the role of high-spin heme b 595 is still a matter of debate. Together with heme d, heme b 595 is proposed to form a binuclear site for the effective reduction of oxygen analogous to the high-spin heme͞ Cu B binuclear center in the heme-copper oxidases (17)(18)(19)(20)(21)(22)(23). The electron transfer sequence in cytochrome bd is thought to be QH 2 3 b 558 3 [b 595 3 d] 3 O 2 (24)(25)(26).…”