Cytochrome Bd-type Oxidase Research Articles

Short-chain aurachin D derivatives are selective inhibitors of E.\xa0coli cytochrome bd-I and bd-II oxidases

Cytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure–activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.

Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus

Gram-positive thermophilic Bacillus species contain cytochrome caa 3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa 3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151–158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of α-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I ( cbdA) encodes 448 amino acid residues with a molecular weight of 50 195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II ( cbdB) encodes 342 residues with a molecular weight of 38 992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a more widely distributed type including the B. stearothermophilus enzyme, suggesting that the latter type is evolutionarily older.

Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry.

A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b595 and b560. The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx. 5 microM). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.

Determination of the oxygen affinities of terminal oxidases in Azotobacter vinelandii using the deoxygenation of oxyleghaemoglobin and oxymyoglobin: cytochrome bd is a low-affinity oxidase

Azotobacter vinelandii is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome o- and bd-types. The latter is required for respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affinities (K m) for oxygen uptake by A. vinelandii cells and membranes respiring dl-malate have been determined by using the deoxygenation of oxyleghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxygen concentration. Dual-wavelength spectrophotometery allowed continuous recording of oxygen consumption over the range 0.003-10 μM, and revealed three distinct affinities for oxygen in a wild-type strain. The kinetic properties of each oxidase were distinguished by the use of two mutants, one lacking and one over-producing the cytochrome bd-type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a high affinity oxidase in all three strains with K m values for membrane preparations of 0.013-0.019 μM. In strains having the cytochrome bd-type oxidase, the K m values measured with intact cells were approximately fourfold higher than in membranes. These results suggest a barrier to the transfer of oxygen to the high affinity component by cytochrome bd, perhaps due to very fast oxygen binding or scavenging by cytochrome d, or to the location of the oxygen-consuming sites of these oxidases on different faces of the membrane. The deoxygenation kinetics of oxymyoglobin revealed the presence of two components with mean K m values of about 0.33 and 4.5 μM. The 4.5 μM component is attributed to the cytochrome bd-type oxidase because it was lacking in intact cells and membranes of the cytochrome bd-deficient mutant strain. The other two components (one with a mean K m value of about 0.33 μM and the highest affinity activity) could not be assigned to particular oxidase(s). The results are interpreted in relation to the physiological role of the cytochrome bd-terminated branch of the respiratory chain and the much higher affinities for oxygen reported for the cytochrome bd-type oxidase in other bacteria.

Oxygen reactions with bacterial oxidases and globins: binding, reduction and regulation

Oxygen is favoured as terminal electron acceptor in aerobic and facultative microorganisms because of its appropriate physical state, satisfactory solubility and its desirable combinations of kinetic and thermodynamic properties. Oxygen is generally reduced by four electrons to yield oxygen, but there are important biological consequences of, and roles for, the partial reduction to superoxide and peroxide. Complex and multiple regulatory networks ensure (i) the utilization of oxygen in preference to other oxidants, (ii) the synthesis of oxygen-consuming enzymes with appropriate properties (particularly affinity for the ligand), and (iii) appropriate cellular protection in the event of oxidative stress. This contribution reviews the terminal respiratory oxidases of selected Gram-negative bacteria and microbial haemoglobin-like proteins. Recent studies of the cytochrome bd-type oxidases of Escherichia coli and Azotobacter vinelandii suggest that, despite probable similarity at the amino acid level, the reactivities of these oxidases with oxygen are strikingly different. The respiratory protection afforded to nitrogenase in the obligately aerobic diazotroph A. vinelandii by the cytochrome bd complex appears to be accompanied by, and may be the result of, a low affinity for oxygen and a high Vmax. The poorly characterized cytochrome o-containing oxidase in this bacterium is not required for respiratory protection. In E. coli, the cytochrome bd-type oxidase has a remarkably high affinity for oxygen, consistent with the view that this is an oxygen-scavenging oxidase utilized under microaerobic conditions. The demonstration of substrate (i.e. oxygen) inhibition in this complex suggests a mechanism whereby wasteful electron flux through a non-proton-pumping oxidase is avoided at higher dissolved oxygen tensions. The demonstration of two ligand-binding sites (haems d and b595) in oxidases of this type suggests plausible mechanisms for this phenomenon. In E. coli, assembly of the cytochrome bd-type oxidase (and of periplasmic cytochromes b and c) requires the presence of an ABC transporter, which may serve to export haem or some "assembly factor" to the periplasm. There is at least one additional oxygen-consuming protein in E. coli-the flavohaemoglobin encoded by the hmp gene. Globin-like proteins are also widely distributed in other bacteria, fungi and protozoa, but most have unknown functions. The function of HMP and the related chimaeric flavohaemoglobins in other bacteria and yeast is unknown; one of several possibilities for HMP is that its relatively low affinity for oxygen during turnover with NADH as substrate could enable it to function as a sensor of failing (or rising) cytoplasmic oxygen concentrations.