Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy.

Marius Horch,Solomon L D Wrathall,Oliver Lenz,Janna Schoknecht,Neil T Hunt,Gregory M Greetham

doi:10.1039/c9sc02851j

Abstract

Hydrogenases are valuable model enzymes for sustainable energy conversion approaches using H2, but rational utilization of these base-metal biocatalysts requires a detailed understanding of the structure and dynamics of their complex active sites. The intrinsic CO and CN- ligands of these metalloenzymes represent ideal chromophores for infrared (IR) spectroscopy, but structural and dynamic insight from conventional IR absorption experiments is limited. Here, we apply ultrafast and two-dimensional (2D) IR spectroscopic techniques, for the first time, to study hydrogenases in detail. Using an O2-tolerant [NiFe] hydrogenase as a model system, we demonstrate that IR pump-probe spectroscopy can explore catalytically relevant ligand bonding by accessing high-lying vibrational states. This ultrafast technique also shows that the protein matrix is influential in vibrational relaxation, which may be relevant for energy dissipation from the active site during fast reaction steps. Further insights into the relevance of the active site environment are provided by 2D-IR spectroscopy, which reveals equilibrium dynamics and structural constraints imposed on the H2-accepting intermediate of [NiFe] hydrogenases. Both techniques offer new strategies for uniquely identifying redox-structural states in complex catalytic mixtures via vibrational quantum beats and 2D-IR off-diagonal peaks. Together, these findings considerably expand the scope of IR spectroscopy in hydrogenase research, and new perspectives for the characterization of these enzymes and other (bio-)organometallic targets are presented.

Highlights

Dihydrogen (H2) is a clean fuel that releases large amounts of free energy but no greenhouse gases upon combustion
Further insights into the relevance of the active site environment are provided by 2D-IR spectroscopy, which reveals equilibrium dynamics and structural constraints imposed on the H2-accepting intermediate of [NiFe] hydrogenases
The Ralstonia eutropha (ReRH) active site produces an IR absorption spectrum containing three peaks (Fig. 1, top) at 1943, 2071, and 2080 cmÀ1 that have been assigned to the fundamental transitions of one CO and two CN stretching vibrations (Fig. 1, bottom; Fig. 2).[4,5,13,16,17,18]

Summary

Introduction

Dihydrogen (H2) is a clean fuel that releases large amounts of free energy but no greenhouse gases upon combustion. Using ultrafast and 2D-IR spectroscopies, we shed light on key bond properties of the CO ligand, dynamic interactions between the diatomic ligands, and the complex interplay between the [NiFe] active site and its protein environment

Results and discussion

Conclusions and outlook

Hydrogen as a Fuel