microbiology, Vol 142, 755-763, Copyright © 1996 by Society for General Microbiology

ARTICLES

The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity in vivo by oxygen inhibition

R D'mello, S Hill and RK Poole
Division of Life Sciences, King's College London, UK.

Cytochrome bd is a respiratory oxidase in Escherichia coli and many other bacteria. It contains cytochromes b558, b595 and d as redox centres, and is thus unrelated to the haem-copper super-family of terminal oxidases. The apparent affinities (Km) for oxygen uptake by respiring cells and membranes from a mutant lacking the alternative oxidase cytochrome bo' were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration. Respiration rates were maximal at oxygen concentrations of 25-50 nM, but the kinetics were complex and indicative of substrate (i.e. oxygen) inhibition. Km values were in the range 3-8 nM (the lowest recorded for a respiratory oxidase), and Ki values between 0.5 and 1.8 microM were obtained. Low temperature photodissociation of anoxic, CO-ligated membranes confirmed the absence of cytochrome bo' and revealed a high-spin b-type cytochrome identified as cytochrome b595 of the cytochrome bd complex. Photodissociation in the presence of oxygen revealed binding of a ligand (presumably oxygen) to cytochrome b595 at a rate much greater than that of CO binding, and formation of the oxygenated form of cytochrome d. The results confirm that both high- spin haems in the cytochrome bd complex bind CO and demonstrate that oxygen can also react with both haems. Substrate inhibition of oxidase activity, in addition to transcriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytochrome bd- and bo'-terminated respiratory pathways.

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