Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

• DOI: 10.1073/pnas.86.20.7823
• Publication Date: 2006-05-31T11:05:49Z
• Authors: M Imai; H Shimada; Y Watanabe; Y Matsushima-Hibiya; R Makino; H Koga; T Horiuchi; Y Ishimura
• Keywords: Alanine

Site-directed mutants of cytochrome P-450cam (the P-450 that acts as the terminal monooxygenase in d-camphor system), which threonine-252 had been changed to alanine, valine, or serine, were employed study role hydroxy amino acid reaction. The mutant enzymes expressed Escherichia coli and purified by a conventional method. All presence exhibited optical absorption spectra almost indistinguishable from those wild-type enzyme their ferric, ferrous, oxygenated, carbon monoxide ferrous forms. In reconstituted system with putidaredoxin its reductase, alanine consumed O2 at rate (1100 per min heme) comparable (1330 heme), whereas amount exo-5-hydroxycamphor formed was less than 10% enzyme. About 85% recovered H2O2. valine also an oxidase activity yield H2O2 accompanied relative decrease activity. On other hand, serine essentially same Thus, uncoupling consumption function produced substitution without hydroxyl group. When binding forms examined, instantaneously oxygenated form, slowly decomposed ferric form rates 5.5 3.2 x 10(-3) sec-1 for former latter enzymes, respectively. Since these too slow account overall consumption, formation considered proceed not way this route but through decomposition peroxide complex reduction reduced putidaredoxin. Based on findings, possible mechanism oxygen activation reaction has discussed.