MS01-01 - Elucidation of NO Reduction Mechanism in Soluble NO Reductase by Time-Resolved Crystallography with Photosensitive Caged Compound

Takehiko Tosha (RIKEN SPring-8 Center, Japan)

Structural determinations of reaction intermediates in metalloenzyme-catalyzed reactions are great challenge in structural biology and very useful for understanding chemistry in metalloenzymes. Recently developed technique called serial femtosecond X-ray crystallography (SFX), in which micro-crystals are continuously supplied to the X-ray irradiation spot, using an X-ray free electron laser facility, SACLA, enabled time-resolved X-ray crystallography (TR-SFX). Since the X-ray laser from SACLA is a ~10 fs pulse, it is possible to carry out a pump-probe type experiment. Here, we applied this method for the observation of enzymatic reaction using photosensitive caged-compound. We utilized a heme-containing enzyme, P450nor, which catalyzes a following reaction; 2NO + NADH + H+→N2O + H2O + NAD+. Photosensitive caged-NO which release two equivalent of NO upon UV irradiation was utilized as a trigger and NO source for the P450nor reaction. We performed TR-SFX at SACLA, and obtained an intermediate structure, substrate NO-bound form, at 20 ms after the UV illumination1). Current results suggest that TR-SFX with photosensitive caged compound is promising approach for the characterization of the reactions catalyzed by metalloenzymes. In the presentation, characterization of next process of the P450nor-catalyzed reaction will be shown.

1. Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged substrate. T. Tosha et al., Nat. Commun. 2017, 8, 1585.