Abstract
Synchrotron-based X-ray structural studies of ligand-bound enzymes are powerful tools to further our understanding of reaction mechanisms. For redox enzymes, it is necessary to study both the oxidized and reduced active sites to fully elucidate the reaction, an objective that is complicated by potential X-ray photoreduction. In the presence of the substrate, this can be exploited to construct a structural movie of the events associated with catalysis. Using the newly developed approach of serial femtosecond rotation crystallography (SF-ROX), an X-ray damage-free structure of the as-isolated copper nitrite reductase (CuNiR) was visualized. The sub-10 fs X-ray pulse length from the SACLA X-ray free-electron laser allowed diffraction data to be collected to 1.6 Å resolution in a 'time-frozen' state. The extremely short duration of the X-ray pulses ensures the capture of data prior to the onset of radiation-induced changes, including radiolysis. Unexpectedly, an O2 ligand was identified bound to the T2Cu in a brand-new binding mode for a diatomic ligand in CuNiRs. The observation of O2 in a time-frozen structure of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from Alcaligenes xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD) since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago.
Highlights
Throughout the 20th century, the increasing use of nitrogen fertilizers to boost food production has resulted in an imbalance of the nitrogen cycle, leading to the accumulation of higher nitrogen oxide levels in soil and surface waters (Gruber & Galloway, 2008; Canfield et al, 2010)
The N-terminal residue modelled as pyroglutamic acid (PCA) in PDB entry 1oe1 has no electron density in this structure and was not built (Ellis et al, 2003)
The global architecture of the structures is near-identical, but the solventderived ligand of the T2Cu was different, with a dioxygen species in the SF-ROX structure in place of the H2O normally observed in SRX structures
Summary
Throughout the 20th century, the increasing use of nitrogen fertilizers to boost food production has resulted in an imbalance of the nitrogen cycle, leading to the accumulation of higher nitrogen oxide levels in soil and surface waters (Gruber & Galloway, 2008; Canfield et al, 2010) Denitrifying microbes utilize these nitrogen oxides as electron acceptors in the anaerobic/aerobic oxidation of organic matter, or less frequently as an inorganic source of electrons. The reduction of nitrite to form NO by nitrite reductase (NiR) is a key step in this process because it is at this point that the loss of terrestrial fixed nitrogen to the atmosphere occurs. The T1Cu site accepts an electron from a carrier protein, either azurin or a c-type cytochrome, and in some CuNiRs these are present in an additional tethered domain (Antonyuk et al, 2013)
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