Activity regulation of photoreactive nitrile hydratase by nitric oxide

作者：Odaka M^*; Fujii K; Hoshino M; Noguchi T; Tsujimura M; Nagashima S; Yohda M; Nagamune T; Inoue Y; Endo I

来源：Journal of the American Chemical Society, 1997, 119(16): 3785-3791.

DOI：10.1021/ja962179c

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a novel enzyme that possesses a non-heme iron(III) center binding endogenous nitric oxide (NO). it is inactivated by aerobic incubation of cells in the dark, whereas the inactive form is converted to the active one by light irradiation. To clarify the mechanism of activity regulation in the NHase, we investigated the role Of NO. When the inactive NHase was irradiated in the Presence of Ee(II) ions and a spin trap, N-methyl-D-glucamine dithiocarbamate (MGD), three hyperfine lines from the nitrogen atom of the [(MGD)(2)-Fe-II-NO] complex were resolved, indicating that MO was released from the enzyme upon photoactivation. The amount of NO release was obtained or 0.99 +/- 0.12 per enzyme (n = 4) by using an NO scavenger, 2-(4-carhoxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. The active NHase was completely inactivated by exogenous NO, and upon photoirradiation 86% of the original activity was restored. FTIR, ESR, and UV-VIS absorption measurements confirmed the view that association of NO restores the original inactive form of thp enzyme. The rate constant for nitrosylation of the enzyme activated by a laser pulse asymptotically increased with an increase in NO concentration. A kinetic analysis of thp NHase nitrosylation demonstrated that inactivation of the enzyme by NO binding proceeded via an intermediate. The rate constant for inactivation and the quantum yield of photoactivation were determined as 14 s(-1) and 0.48, respectively. It is thus concluded that the activity of the NHase is regulated by nitrosylation and photoinduced denitrosylation of the non-heme iron, center. This finding provides anew aspect regarding biological function of NO, i.e., regulation of the enzymatic activity with the aid of light.

• 出版日期1997-4-23