A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] Hydrogenase

Abstract

Kinetic, EPR, and Fourier transform infrared spectroscopic analysis of Desulfovibrio fructosovorans [NiFe]hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H 2 /ortho-H 2 conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover numbers for the oxidation of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H 2 oxidation and hydrogen isotope exchange activities, but did not change the para-H 2 /ortho-H 2 conversion activity. The spectroscopic properties of this mutant were totally different, especially in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.Many microorganisms use molecular hydrogen in their metabolic routes as an energy source or for evacuating an excess of electrons. The enzymes that catalyze reversibly the conversion of molecular hydrogen to two electrons and two protons are known as hydrogenases. Although this is the simplest chemical reaction, the catalytic mechanism of these enzymes is quite complicated, and its details are still a matter of debate (1). Hydrogen isotope exchange experiments indicate that the H 2 cleavage reaction is heterolytic; thus, a hydride and a proton are formed in the first step (2, 3). In the second step, the two electrons of the hydride are extracted, and a second proton is formed. Subsequently, the two electrons have to be transported, via the intramolecular electron transfer chain, from the active site to the redox partner of the hydrogenase (a redox protein or NAD ϩ (P)) in vivo, or a redox dye in vitro; the two protons have to be transferred to the protein environment as well. These steps are reversed in the case of H 2 production activity (1).How do all these steps take place in hydrogenases? These proteins are metalloenzymes that all contain iron, and in many cases, also nickel. The crystallographic structures of several [Fe] hydrogenases (4, 5) and [NiFe] hydrogenases (6, 7) have been obtained by x-ray diffraction studies. In both types of enzymes, the active site is a deeply buried bimetallic center, in which the metals are bridged by thiol groups and have CO and CN Ϫ as ligands. This type of coordination favors the binding of molecular hydrogen or hydride to the active site (1, 8). The crystal structures also indicate that Fe-S clusters are located between the active site and the protein surface, which are thought to form the intramolecular electron pathway in the H 2 production/oxidation mechanism (9). In [NiFe] hydrogenases, one nickel and one iron atom form the bimetallic center. The nickel is coordinated to four cysteine ligands via their thiol groups. Two of them are terminal ligands, and the other...

Experimental approaches to kinetics of gas diffusion in hydrogenase

“…In the oxidized as-prepared state, both mutants exhibit spectra similar to those reported for the WT enzyme (22). The oxidized active site normally gives a superposition of signals called Ni-A (g ϭ 2.31, 2.23, 2.01) and Ni-B (g ϭ 2.34, 2.16, 2.01).…”

“…The assays of H2 oxidation were carried out after the enzymes were activated (22). The isotope-exchange assay is described in ref.…”

“…The D. fructosovorans and Escherichia coli strains, plasmids, growth conditions, and site-directed mutagenesis methods used in this study are described in ref. 22. The Strep tag II sequence (IBA) was introduced at the 5Ј terminus of the large subunit gene (hynB).…”

Mechanism of Proton Transfer in [FeFe]-Hydrogenase from Clostridium pasteurianum

“…In previous work, it has been observed that proton exchange within proteins can be facilitated via negatively charged side chains and water molecules (25)(26)(27). Understand-ing this key mechanism in [FeFe]-hydrogenases is critical for targeted protein engineering, as perturbing proton transfer can cause unintended deleterious effects on the catalytic rate (28). To date, detailed experiments investigating proton transport within [FeFe]-hydrogenases have not been reported.…”

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

“…It has been proposed that the enzyme interconverts between NiSI and NiR when catalyzing HDE (20,21). A combination of these three assays has been used to investigate PT in hydrogenase (22,23); for example, if a certain variant is affected in both H 2 production/oxidation and H/D exchange, but catalyzes the conversion between para and ortho H 2 , then it is likely that the mutated residue is involved in PT. This approach was used to show that Glu-25 L in the large subunit of the enzyme from Df and Glu-13 L in the sensor enzyme from Ralstonia eutropha are essential for PT (22,23).…”