Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase

A sub-ångström-resolution X-ray crystal structure of [NiFe] hydrogenase, with direct detection of the products of the heterolytic splitting of dihydrogen into a hydride bridging the Ni and Fe and a proton attached to the sulphur of a cysteine ligand. Hydrogens visualized in hydrogenase enzyme [NiFe] hydrogenases use nickel and iron to catalyse the reversible oxidation of molecular hydrogen. They are the focus of much research worldwide because of their potential in biotechnology and in serving as natural models for biomimetic catalysts in the energy sector for hydrogen production and conversion. In protein X-ray crystallography it is notoriously difficult to detect hydrogens, a particularly significant problem in hydrogenases where hydrogens are involved directly in the reaction. Hideaki Ogata et al. have succeeded in obtaining a sub-ångström resolution X-ray crystal structure of [NiFe] hydrogenase leading to detection of most of the hydrogens even close to the metal ions. Using their technique authors were able to detect the products of the heterolytic splitting of dihydrogen: a hydride that bridges the Ni and Fe ions, and a proton that is attached to the sulfur of a cysteine ligand. The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst.sup.1. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein.sup.2,3,4,5,6. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution.sup.7. Here we report the crystal structure of a standard [NiFe] hydrogenase (~91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H.sup.-) bridging the Ni and Fe and a proton (H.sup.+) attached to the sulphur of a cysteine ligand. The Ni-H.sup.- and Fe-H.sup.- bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN.sup.- ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
Author(s): Hideaki Ogata [sup.1] , Koji Nishikawa [sup.1] [sup.2] , Wolfgang Lubitz [sup.1] Author Affiliations: (1) Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr, [...]