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

The cytochrome bd ubiquinol oxidase from Escherichia coli is induced when the bacteria are cultured under microaerophilic or low-aeration conditions. This membrane-bound respiratory oxidase catalyses the two-electron oxidation of ubiquinol and the four-electron reduction of dioxygen 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 it is likely that haem d and b(595) form a bimetallic site in the enzyme. 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 associated with the quinol oxidation site, which has also been shown to be within subunit I. In a previous effort to locate the specific amino acids axially ligated to haem b(558), all six histidines within subunit I were 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 current work, a combination of low-temperature near-infrared magnetic circular dichroism (NIR-MCD) and EPR spectroscopies have been employed to identify the nature of the haem b(558) axial ligands. The NIR-MCD spectrum 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 strong evidence for histidine-methionine ligation. It is concluded that haem b(558) is ligated to histidine-186 plus one of the methionines located within subunit I of the oxidase.