Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography.

Thomas P Halsted,Hideo Ago,S Samar Hasnain,Kunio Hirata,Chai C Gopalasingam,Masaki Yamamoto,Svetlana V Antonyuk,Keitaro Yamashita,Matthew P Blakeley,Robert R Eady,Go Ueno,Rajesh T Shenoy

doi:10.1107/s2052252519008285

Abstract

Copper-containing nitrite reductases (CuNiRs) that convert NO2 - to NO via a CuCAT-His-Cys-CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD-, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2 - a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.

Highlights

The highly brilliant undulator beamlines at modern synchrotron facilities have facilitated the structure determination of biological molecules and their complexes at high resolution using conventional synchrotron-radiation crystallography (SRX)
The plasmid was transformed into E. coli BL21 (DE3) cells via heat shock and the transformant was cultured on KanR lysogeny broth (LB) agar to isolate individual colonies. 500 ml LB supplemented with 30 mg mlÀ1 kanamycin was inoculated with a single colony and was incubated with shaking at 37C
Resting-state structures of Achromobacter cycloclastes CuNiR (AcNiR) determined by SF-ROX and neutron crystallography

Summary

Introduction

The highly brilliant undulator beamlines at modern synchrotron facilities have facilitated the structure determination of biological molecules and their complexes at high resolution using conventional synchrotron-radiation crystallography (SRX). The brilliance of the X-rays at some of the state-ofthe-art crystallographic beamlines has enabled this to be achieved using much smaller (10–30 mm) crystals than was anticipated at the turn of the century These gains have come at the expense of an increased absorbed X-ray dose per unit volume and the potential for concomitant radiolysis and radiation damage (Garman, 2010; Yano et al, 2005; Horrell et al, 2016). The recent advent of XFEL crystallography using femtosecond X-ray pulses provides a new opportunity to obtain damage-free structures (Suga et al, 2014, 2017), adding to neutron crystallography and NMR, which have remained the only radiationdamage-free structural probes for decades, albeit with their own particular limitations (Blakeley et al, 2015; Blakeley, 2009; Luchinat & Banci, 2017)

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