Oscar Gutiérrez-Sanz

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.

Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants.

Nickel-containing hydrogenases, the biological catalysts of oxidation and production, reversibly inactivate under anaerobic, oxidizing conditions. We aim at understanding the mechanism of (in)activation and what determines its kinetics, because there is a correlation between fast reductive reactivation and oxygen tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnology. Direct electrochemistry is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here we describe simple analytical models that we used to characterize and compare 16 mutants, obtained by substituting the position-74 valine of the -sensitive NiFe hydrogenase from Desulfovibrio fructosovorans. We observed that this substitution can accelerate reactivation up to 1,000-fold, depending on the polarity of the position 74 amino acid side chain. In terms of kinetics of anaerobic (in)activation and oxygen tolerance, the valine-to-histidine mutation has the most spectacular effect: The V74H mutant compares favorably with the -tolerant hydrogenase from Aquifex aeolicus, which we use here as a benchmark.

Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states

Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni–Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-‘Sox’ species.

The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis

Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 production. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

Influence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenases

A combined experimental and theoretical study of the catalytic activity of a [NiFeSe] hydrogenase has been performed by H/D exchange mass spectrometry and molecular dynamics simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or production of H2. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the nickel atom in the active site instead of cysteine. The aim of this research is to determine how much the specific catalytic properties of this hydrogenase are influenced by the replacement of a sulfur by selenium in the coordination of the bimetallic active site versus the changes in the protein structure surrounding the active site. The pH dependence of the D2/H+ exchange activity and the high isotope effect observed in the Michaelis constant for the dihydrogen substrate and in the single exchange/double exchange ratio suggest that a “cage effect” due to the protein structure surrounding the active site is modulating the enzymatic catalysis. This “cage effect” is supported by molecular dynamics simulations of the diffusion of H2 and D2 from the outside to the inside of the protein, which show different accumulation of these substrates in a cavity next to the active site.

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

Abstract ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0‐ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode‐assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.

A Threonine Stabilizes the Nic and Nir Catalytic Intermediates of [Nife]-Hydrogenase.

Background: A conserved threonine in [NiFe]-hydrogenases is a putative proton transfer relay. Results: Poorly active variants have modified spectroscopic signatures associated with changes in local protein structure. Conclusion: This threonine is not necessarily a proton transfer relay but, rather, stabilizes reaction intermediates. Significance: Combined kinetic, spectroscopic, and structural characterizations of several variants are necessary to assess the role of a residue in [NiFe]-hydrogenase. The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

Induction of a Proton Gradient across a Gold‐Supported Biomimetic Membrane by Electroenzymatic H2 Oxidation

Energy-transduction mechanisms in living organisms, such as photosynthesis and respiration, store light and chemical energy in the form of an electrochemical gradient created across a lipid bilayer. Herein we show that the proton concentration at an electrode/phospholipid-bilayer interface can be controlled and monitored electrochemically by immobilizing a membrane-bound hydrogenase. Thus, the energy derived from the electroenzymatic oxidation of H2 can be used to generate a proton gradient across the supported biomimetic membrane.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.