THE CYTOCHROME BD QUINOL OXIDASE IN ESCHERICHIA-COLI HAS AN EXTREMELYHIGH OXYGEN-AFFINITY AND 2 OXYGEN-BINDING HEMES - IMPLICATIONS FOR REGULATION OF ACTIVITY IN-VIVO BY OXYGEN INHIBITION

Cytochrome bd is a respiratory oxidase in Escherichia coli and many ot her bacteria, It contains cytochromes b(558), b(595) and d as redox ce ntres, and is thus unrelated to the haem-copper super-family of termin al oxidases. The apparent affinities (K-m) for oxygen uptake by respir ing cells and membranes from a mutant lacking the alternative oxidase cytochrome bo' were determined by deoxygenation of oxyleghaemoglobin a s a sensitive reporter of dissolved oxygen concentration. Respiration rates were maximal at oxygen concentrations of 25-50 nM, but the kinet ics were complex and indicative of substrate (i.e. oxygen) inhibition. K-m values were in the range 3-8 nM (the lowest recorded for a respir atory oxidase), and K-i values between 0.5 and 1.8 mu M were obtained. Low temperature photodissociation of anoxic, CO-ligated membranes con firmed the absence of cytochrome bo' and revealed a high-spin b-type c ytochrome identified as cytochrome b(595) of the cytochrome bd complex . Photodissociation in the presence of oxygen revealed binding of a li gand (presumably oxygen) to cytochrome b(595) at a rate much greater t han that of CO binding, and formation of the oxygenated form of cytoch rome d. The results confirm that both high-spin haems in the cytochrom e bd complex bind CO and demonstrate that oxygen can also react with b oth haems. Substrate inhibition of oxidase activity, in addition to tr anscriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytoch rome bd- and bo'-terminated respiratory pathways.