New horizons in structure-function studies of copper nitrite reductase

Abstract

In recent years there have been paradigm shifts in our understanding of the diversity of copper-containing nitrite reductases (CuNiRs) found in nature, as genomic mining revealed the widespread distribution of variants with additional tethered redox domains. The availability of these variants is an unparalled opportunity to explore inter-protein electron transfer events that are so important in biology. Access to X-ray free-electron laser sources with femtosecond X-ray pulses has enabled crystal structures of several CuNiRs to be determined free of radiation-induced damage and radiation-induced redox chemistry. By controlling the radiation dose, structural movies of enzyme-bound intermediates of the catalytic cycle have been identified, notably the relevance of a Cu-NO species to catalysis has been established. Such studies have been complemented by an approach using chemically induced turnover of enzyme in crystals. These have allowed damage-free high-resolution, even atomic resolution, structures to be analysed to assign probable protonation states of critical residues complemented by ambient temperature and our neutron crystallographic structure. The determination of X-ray crystal structures at elevated temperatures including room temperature revealed a stable Cu-nitrosyl intermediate during in-crystal turnover. This should enable refinement in computational chemistry modelling and enable a more rational comparison with spectroscopic data relating to solution studies. Using advanced biophysical techniques the link between proton uptake and inter-Cu electron transfer been established. Spectroscopic single-molecule studies have revealed differences in redox behaviour of isolated enzyme compared with turnover. These are significant advances that together with recent refinement of computational models of the CuNiR reaction mechanism are likely to assist in the development of synthetic CuNiRs.