QM/MM Simulations of Protein Crystal Reactivity Guided by MSOX Crystallography: A Copper Nitrite Reductase Case Study.

Kakali Sen,Chin Yong,Michael A. Hough,Richard W. Strange,Thomas W. Keal

doi:10.1021/acs.jpcb.1c03661

Abstract

The recently developed multiple structures from one crystal (MSOX) serial crystallography method can be used to provide multiple snapshots of the progress of enzymatic reactions taking place within a protein crystal. Such MSOX snapshots can be used as a reference for combined quantum mechanical/molecular mechanical (QM/MM) simulations of enzyme reactivity within the crystal. QM/MM calculations are used to identify details of reference states that cannot be directly observed by X-ray diffraction experiments, such as protonation and oxidation states. These reference states are then used as known fixed endpoints for the modeling of reaction paths. We investigate the mechanism of nitrite reduction in an Achromobacter cycloclastes copper nitrite reductase crystal using MSOX-guided QM/MM calculations, identifying the change in nitrite binding orientation with a change in copper oxidation state, and determining the reaction path to the final NO-bound MSOX structure. The results are compared with QM/MM simulations performed in a solvated environment.

Highlights

Macromolecular crystal structures are usually obtained as single, static structures from one or more crystals, representing the average structure over the time duration of the experiment and over the X-ray exposed region of the crystal
Guided by the MSOX data series, quantum mechanical/molecular mechanical (QM/MM) calculations unambiguously assign the Cu(II) oxidation state to the initial top-hat orientation of nitrite bound to T2Cu, and Cu(I) to the subsequent side-on orientated structure, in line with previous density functional theory (DFT) calculations on model complexes
The QM/MM calculations further indicate that the bound species at both points is NO2− rather than HNO2

Summary

Introduction

Macromolecular crystal structures are usually obtained as single, static structures from one or more crystals, representing the average structure over the time duration of the experiment and over the X-ray exposed region of the crystal. The multiple structures from one crystal (MSOX) approach differs from “standard” crystallographic methods in that a series (tens to hundreds) of complete X-ray diffraction data sets are measured consecutively from the same volume of just one protein crystal.[1,2] Each exposure of the crystal to the X-ray beam produces a large number of solvated photoelectrons[3] from radiolysis of water molecules within the crystal, providing a source of reducing power to initiate catalytic reactions or other changes in the crystalline enzyme In this manner, subsequent data sets from the dose series represent later stages of redox-driven reactions. This approach relies on site-specific (e.g., redox) changes to the enzyme at the metal-containing active sites occurring at far lower absorbed Xray doses than those which cause “global” radiation damage such as loss of diffracting power.[9−11] For example, we recently collected a series of 75 data sets (resolution 1.08−1.84 Å) at 190 K from one crystal of Achromobacter cycloclastes copper nitrite reductase (AcNiR) and a series of 20 data sets (resolution 1.4−1.9 Å) at room temperaturea from a similar

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