Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions.
Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase. Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde. We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase. Because E. coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes. We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D. We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase. Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112-0850, USA.
Kenneth R
KR
Brown
Brienne M
BM
Allan
Peter
P
Do
Eric L
EL
Hegg
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D005294:Ferrochelatase / Q000737:chemistry* / Q000235:genetics; D005798:Genes, Bacterial; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010100:Oxygen / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C069808:heme O; C027728:heme a; D006418:Heme; D010105:Oxygenases; D019883:Alkyl and Aryl Transferases; C078356:CyoE protein, Bacteria; C489727:CyoE protein, E coli; D005294:Ferrochelatase; D010100:Oxygen