Marc Rousset

Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzymes selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

Experimental approaches to kinetics of gas diffusion in hydrogenase

Hydrogenases, which catalyze H2 to H+ conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.

Introduction of Methionines in the Gas Channel Makes [NiFe] Hydrogenase Aero-Tolerant

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Reduction of Technetium(VII) by Desulfovibrio fructosovorans Is Mediated by the Nickel-Iron Hydrogenase

ABSTRACT Resting cells of the sulfate-reducing bacteriumDesulfovibrio fructosovorans grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase. The role of cytochromec3 in the mechanism is also discussed.

Original design of an oxygen-tolerant [NiFe] hydrogenase: major effect of a valine-to-cysteine mutation near the active site.

Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.

Understanding and Tuning the Catalytic Bias of Hydrogenase

When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H(2) oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site.

O2-independent formation of the inactive states of NiFe hydrogenase

We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.

Shewanella oneidensis : a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

BackgroundThe eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.ResultsBased on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB) transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.ConclusionIn the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans

A strain of Desulfovibrio fructosovorans deleted from the hydN [NiFe]hydrogenase structural gene was constructed. A plasmid carrying a 7 kb DNA fragment on which the hydN gene had been replaced by the npt reporter gene (kanamycin‐resistant, KnR) was introduced into D. fructosovorans by electroporation. Southern analysis of one KnR clone demonstrated that the hydN gene had been eliminated by marker exchange. This mutant, which was devoid of the [NiFe]hydrogenase gene, still showed a 10% residual hydrogenase activity. Its ability to grow efficiently with H2 as sole energy source is discussed. This is the first report, in a member of the sulphate‐reducing bacteria, of a successful transformation and concomitant homologous recombination leading to a fully controlled genotype.

Cloning and sequencing of the locus encoding the large and small subunit genes of the periplasmic [NiFe]hydrogenase from Desulfovibrio fructosovorans

The genetic locus encoding the periplasmic [NiFe]hydrogenase (Hyd) from Desulfovibrio fructosovorans was cloned and sequenced. The genes of this two-subunit enzyme have an operon organization in which the 0.94-kb gene encoding the small subunit precedes the 1.69-kb gene encoding the large subunit. A Shine-Dalgarno sequence is centered at -9 bp from the ATG of both subunits. The possible presence of another open reading frame downstream from the large-subunit-encoding gene is considered. The N-terminal sequence of the large 61-kDa subunit deduced from the nucleotide sequence is in perfect agreement with the results of the amino acid (aa) sequence determined by Edman degradation. A 50-aa leader peptide precedes the small 28-kDa subunit. The aa sequence of the enzyme shows nearly 65% homology with the [NiFe]Hyd aa sequence of Desulfovibrio gigas. Comparisons with a large range of Hyds from various bacterial species indicate the presence of highly conserved Cys residues, the implications of which are discussed from the point of view of nickel atom and cluster accommodation.