Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography

Abstract

Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

“…S12). Moreover, we recently showed that the imidazole ring of His244 in GtNiR, which corresponds to His255 in AfNiR, rotates as a result of photoreduction, but not the difference of temperatures (55). Therefore, cryogenic temperature would not be the only factor for the rotation and the reduction of Cu may also cause it, as was predicted previously (15).…”

Recent structural insights into the function of copper nitrite reductases

“…Little to no change to the overall fold of the enzyme or in the vicinity of the T1Cu site were seen throughout the MSOX series. The low dose atomic resolution starting structure exhibited a previously unseen dual conformation of NO 2 À , in ''top-hat'' and ''side-on'' bidentate orientations, of which the top-hat conformation matches the vertical substrate coordination from 'radiation-damagefree' XFEL structures, 54,58 corroborating the idea that this vertical binding mode represents the initial nitrite binding position. Overall, the 45-dataset MSOX series demonstrated several structural changes at the T2Cu site, Fig.…”

“…27 Most recently this phenomenon has been exploited to drive in crystallo reactions to generate 'structural movies' of enzyme catalysis in green AcNiR 55,57 and thermostable Geobacillus thermodenitrificans (GtNiR). 58 T2Cu site mutations in AfNiR significantly altered the binding mode of nitrite 21 and these were related to changes in enzyme activity. Mutation of this Ile CAT to Val or Leu led to asymmetric bidentate NO 2 À binding while mutation to Met, Thr, Gly or Ala resulted in monodentate coordination, allowing a water molecule to also bind to T2Cu in a position close to that usually occupied by the O2 atom of nitrite.…”

“…85 The first CuNiR XFEL structures at room temperature were recently obtained using the Spring-8 Compact Free Electron Laser (SACLA). 54,58 Comparison of the 1.43 Å resolution serial femtosecond crystallography (SFX) structure of resting state GtNiR 58 with conventional synchrotron radiation (SRX) structures revealed a 101 rotation of the imidazole ring of His CAT , in the SRX data in relation to the SFX structure. This led to the suggestion that His CAT may function as a proton-relay switch, whereby T2Cu reduction causes His CAT rotation thus altering the H-bonding network of His CAT with Thr268 and Glu267 to destabilise the positive charge of His CAT and facilitate proton transfer to the bridging water.…”

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

“…Using high photon density in the focused XFEL beam, which achieves single-pulse diffraction within femtosecond exposure time, SFX enables protein structure determination from micrometer-to submicrometer-sized crystals at ambient temperature. The femtosecond pulse duration allows the "diffraction-beforedestruction" approach by circumventing radiation damage of the sample, because the diffraction process can be terminated on a timescale shorter than that of the damage process (4)(5)(6). Moreover, SFX has become a powerful tool for time-resolved analyses of light-driven structural changes and chemical dynamics (7)(8)(9).…”