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PMID:12206660
| Citation |
Brown, KR, Allan, BM, Do, P and Hegg, EL (2002) Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions. Biochemistry 41:10906-13 |
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| Abstract |
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. |
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| Keywords |
Alkyl and Aryl Transferases/chemistry; Alkyl and Aryl Transferases/genetics; Animals; Bacillus subtilis/enzymology; Bacillus subtilis/genetics; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Cattle; Cloning, Molecular; Escherichia coli Proteins; Ferrochelatase/chemistry; Ferrochelatase/genetics; Genes, Bacterial; Heme/analogs & derivatives; Heme/chemistry; Heme/genetics; Heme/isolation & purification; Oxygen/chemistry; Oxygenases/chemistry; Spectrometry, Mass, Electrospray Ionization |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0004497: monooxygenase activity |
ECO:0000269: |
F |
Scheme 3. "The first step produces the alcohol intermediate, while the second step forms the corresponding geminal diol. Spontaneous dehydration of the diol leads to heme A, while the overoxidized carboxylate byproduct arises from a third reaction. This process shows CtaA oxidizes heme O to heme A via monooxygenase reaction. |
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