THE HEME B(558) COMPONENT OF THE CYTOCHROME BD QUINOL OXIDASE COMPLEXFROM ESCHERICHIA-COLI HAS HISTIDINE METHIONINE AXIAL LIGATION

The cytochrome bd ubiquinol oxidase from Escherichia coli is induced w hen the bacteria are cultured under microaerophilic or low-aeration co nditions. This membrane-bound respiratory oxidase catalyses the two-el ectron oxidation of ubiquinol and the four-electron reduction of dioxy gen to water. The oxidase contains three haem prosthetic groups: haem b(558), haem b(595) and haem d Haem dis the oxygen binding site, and i t is likely that haem d and b(595) form a bimetallic site in the enzym e. Haem b(558) has been previously characterized spectroscopically as being low spin and has been shown to be located within subunit I(CydA) of this two-subunit enzyme. It is likely that haem b(558) is associat ed with the quinol oxidation site, which has also been shown to be wit hin subunit I. In a previous effort to locate the specific amino acids axially ligated to haem b(558), all six histidines within subunit I w ere altered by site-directed mutagenesis. Only one, histidine-186, was identified as a likely ligand to haem b(558). Hence it was suggested that haem b(558) could not have bis(histidine) ligation. In the curren t work, a combination of low-temperature near-infrared magnetic circul ar dichroism (NIR-MCD) and EPR spectroscopies have been employed to id entify the nature of the haem b(558) axial ligands. The NIR-MCD spectr um at cryogenic temperatures is dominated by the low-spin haem b(558) component of the complex, and the low-energy band near 1800 nM is stro ng evidence for histidine-methionine ligation. It is concluded that ha em b(558) is ligated to histidine-186 plus one of the methionines loca ted within subunit I of the oxidase.