Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

Abstract

An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.

Figure 1

Complementary methods for metalloprotein serial crystallography. Clockwise from top left. UV–vis absorption spectra of a B-type dye decolorizing peroxidase (electron density shown centre) [9]. Oxidation states of iron sites in a ferredoxin were revealed using diffraction spectroscopy as shown through the use of simulated diffraction patterns [∗∗36]. Iron Kα XES of methane monooxygenase (MMOH) crystals confirming iron oxidation state [∗33]. Reprinted with permission from Ref. [33]. Copyright 2020 American Chemical Society. Single crystal resonance Raman spectra of an A-type dye-decolorizing peroxidase confirming photoreduction of the heme group during X-ray data collection [47]. Images from Refs. [9,36,47] reproduced with permission from the International Union of Crystallography.

Figure 2

X-ray and light-driven dynamics in metalloproteins. Top: Following X-ray induced changes in a dye-type heme peroxidase crystal using SSX. (a) While differences between synchrotron and XFEL structures are well resolved for high dose SSX structures of DtpAa (a), X-ray-driven displacement of water is apparent even at low doses, which a dose-series reveal to be the first step of an X-ray-driven migration (b) [8]. Reproduced with permission from the International Union of Crystallography. Bottom: SFX structures of P450nor with use of a photocage to obtain intact structures of catalytic intermediates free from radiation damage. (c) Resting-state structure and (d) transient structure 20 ms after caged-NO photolysis in the absence of NADH [∗44]. Reproduced with permission from the Springer Nature.