Abstract
Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07-1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a 'catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.
Highlights
A major objective in structural biology is to determine accurate structures of all relevant intermediates in an enzyme mechanism. This ambition has only been partially fulfilled in most cases owing to difficulties in obtaining crystals in the relevant reaction states or to differences between individual crystals, as well as the deleterious effects of X-ray radiation damage/radiolysis, which has long been a concern in structural biology (Ravelli & Garman, 2006; Garman, 2010; Garman & Weik, 2015)
Only one crystal diffracting to atomic resolution was used to probe the reaction mechanism of CuNiRs by exploiting the ability of X-ray-generated solvated electrons to drive the catalytic reaction
These challenges are likely to be addressed in the future, where developments in automated data handling and model building are being driven by highthroughput serial crystallography using X-ray free-electron lasers (XFELs) and nextgeneration higher brightness synchrotron-radiation sources (e.g. ESRF Phase II and MAX IV; Borland, 2013; Eriksson et al, 2014) and detectors
Summary
A major objective in structural biology is to determine accurate structures of all relevant intermediates in an enzyme mechanism. Only relatively recently has it become widely recognized that the reduction of redox centres by X-ray-generated photoelectrons can occur very rapidly, often prior to significant loss of diffraction resolution, damage to disulfide bridges, thiols or amino-acid side chains (Beitlich et al, 2007; Antonyuk & Hough, 2011; Yano et al, 2005). Such X-ray-induced changes to the redox states of metal centres can lead to mis-assignment of functional states or steps in a catalytic mechanism to a particular crystal structure. This approach has more recently been used to characterize X-raygenerated species in, for example, tobacco assimilatory nitrite reductase (Nakano et al, 2012; an Fe–S cluster- and sirohaemcontaining protein that converts nitrite to ammonium), horseradish peroxidase and multicopper oxidases (Berglund et al, 2002; Komori et al, 2014; De la Mora et al, 2012)
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