Heme A Synthase Does Not Incorporate Molecular Oxygen into the Formyl Group of Heme A

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 ring D to an aldehyde, a reaction catalyzed by heme A synthase. To effect this transformation, heme A synthase is 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 on molecular oxygen. Surprisingly, when cells expressing heme A synthase were incubated with (18)O(2), no significant incorporation of label was observed in heme A, the C8 alcohol intermediate, or the C8 overoxidized byproduct. Conversely, when the cells were grown in H(2)(18)O, partial labeling was observed at every heme oxygen position. These results suggest that the oxygen on the heme A aldehyde is derived from water. Although our data do not allow us to exclude the possibility of exchange with water inside of the cell, the results seem to question a mechanism utilizing successive monooxygenase reactions and support instead a mechanism of heme O oxidation via electron transfer.

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…Additionally, in heme a, a methyl group at carbon 8 is oxidized to an aldehyde. Heme o is the substrate for this modification, which is catalyzed by a heme-containing monooxygenase (or peroxidase) enzyme called heme a synthase, encoded by the Bacillus ctaA and the related yeast COX11 genes (31,32,104,(149)(150)(151). In vivo, the heme o and a synthases may form a complex for substrate channeling (30).…”

“…One is a b heme that transfers electrons in the oxidative reaction, and the other is the heme o substrate (32,(149)(150)(151). Since the conserved histidines are at the outer surface of the membrane, it is suggested that the heme b substrate for CyoE (CtaB) and the heme o substrate for CtaA may be located on opposite sides of the inner membrane.…”

Heme A Synthase Enzyme Functions Dissected by Mutagenesis ofBacillus subtilisCtaA

“…The second hydroxylation would result in an unstable dihydroxymethyl group, which spontaneously decomposes into a formyl group and a water molecule. Isotope labeling experiments suggest that the oxygen atom of the formyl group is not directly derived from molecular oxygen (4). This is unexpected, and notably, B. subtilis heme A synthase does not have the biochemical or amino acid sequence characteristics of a P450 type of enzyme, which contains heme as a prosthetic group and activates molecular oxygen (25).…”

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b

“…1). This suggested a more complicated reaction mechanism where, like in hemesidechain-modifying enzymes (3,4) or cyclooxygenases (10), electrons or protons might be conveyed from the substrate to a reactive Fe intermediate via a redox-active amino acid side chain. Such transfers of protons and electrons could occur sequentially or by proton-coupled electron transfer (PCET).…”

“…Such transfers of protons and electrons could occur sequentially or by proton-coupled electron transfer (PCET). 3 Alternatively, homolytic scission of an Fe(III)coproheme-OOH bond could yield a hydroxyl radical (•OH) that is channeled by the active site toward a specific C-H bond on the reactive propionate. A mechanism of this type would be consistent with the proposed selfhydroxylation catalyzed by heme oxygenases (1), and with the decarboxylase structure (9), which lacks the typical apparatus of enzymes that activate H2O2 by heterolytic cleavage.…”