Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center.

Recently, we discovered an intriguing hemoprotein [aliphatic aldoxime dehydratase (OxdA)] that catalyzes the dehydration of aliphatic aldoximes [R-CH=N-OH] to the corresponding nitriles [R-C identical withN] in the industrial Pseudomonas chlororaphis B23 strain. Unlike the utilization of H(2)O(2) or O(2) as a mediator of the catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron, experimental evidence of which was obtained here by means of resonance Raman (RR) analysis with an isotope technique. We found that the addition of a large amount of butyraldoxime (final concentration, 200 mM) to ferrous OxdA with a low enzyme concentration (final concentration, 5 muM) yields a long-lived OxdA-substrate complex (named OS-II), whose UV-vis spectrum is different from the corresponding spectra of the OxdA-substrate complex I and CO-bound, ferrous, and ferric forms of OxdA. Intriguingly, the RR analysis demonstrated that OS-II includes a highly oxidized heme with strong bonding between a substrate and the heme iron, as judged from the heme oxidation state marker nu(4) band at 1,379 cm(-1) and the (15)N-isotope-substituted butyraldoxime sensitive band at 857 cm(-1) in the RR spectra. It is noteworthy that OS-II has a highly oxidized heme like the ferryl-oxo heme species (e.g., compound II) formed by some general hemoproteins, although the function of OxdA is different from those (transport of electrons, transport of oxygen, sensing of oxygen or carbon monoxide, and catalysis of redox reactions) of general hemoproteins.