| Abstract
| - Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidases.The final step in heme A biosynthesis requires the oxidation of the C8 methyl substituent on pyrrole ringD to an aldehyde, a reaction catalyzed by heme A synthase. To effect this transformation, heme A synthaseis proposed to utilize a heme B cofactor, oxidizing the substrate via successive monooxygenase reactions.Consistent with this hypothesis, the activity of heme A synthase is found to be strictly dependent onmolecular oxygen. Surprisingly, when cells expressing heme A synthase were incubated with 18O2, nosignificant incorporation of label was observed in heme A, the C8 alcohol intermediate, or the C8overoxidized byproduct. Conversely, when the cells were grown in H218O, partial labeling was observedat every heme oxygen position. These results suggest that the oxygen on the heme A aldehyde is derivedfrom water. Although our data do not allow us to exclude the possibility of exchange with water insideof the cell, the results seem to question a mechanism utilizing successive monooxygenase reactions andsupport instead a mechanism of heme O oxidation via electron transfer.
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