Christine Cavazza

Visible Light-Driven H2 Production by Hydrogenases Attached to Dye-Sensitized TiO2 Nanoparticles

A study of hybrid, enzyme-modified nanoparticles able to produce H(2) using visible light as the energy source has been carried out to establish per-site performance standards for H(2) production catalysts able to operate under ambient conditions. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) is identified as a particularly proficient catalyst. The optimized system consisting of Db [NiFeSe]-H attached to Ru dye-sensitized TiO(2), with triethanolamine as a sacrificial electron donor, produces H(2) at a turnover frequency of approximately 50 (mol H(2)) s(-1) (mol total hydrogenase)(-1) at pH 7 and 25 degrees C, even under the typical solar irradiation of a northern European sky. The system shows high electrocatalytic stability not only under anaerobic conditions but also after prolonged exposure to air, thus making it sufficiently robust for benchtop applications.

Fe-only hydrogenases: structure, function and evolution

Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons according to the reversible reaction: H(2)<==>2H(+)+2e(-). Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases. Extensive spectroscopic, electrochemical and structural studies have shed appreciable light on the catalytic mechanism of hydrogenases. Although evolutionarily unrelated, NiFe and Fe-hydrogenases share a common, unusual feature: an active site low-spin Fe center with CO and CN coordination. We have recently focused our attention on Fe-hydrogenases because from structural studies by us and others, it appears to be a simpler system than the NiFe counterpart. Thus the primary hydrogen binding site has been identified and plausible, electron, proton and hydrogen pathways from and to the buried active site may be proposed from the structural data. The extensive genome sequencing effort currently under way has shown that eukaryotic organisms contain putatively gene coding sequences that display significant homology to Fe-hydrogenases. Here, we summarize the available evidence concerning the mechanism of these enzymes and carry out a structural comparison between Fe-hydrogenases and related proteins of unknown metal content from yeast, plant, worm, insect and mammals.

Structure-function relationships of anaerobic gas-processing metalloenzymes

Reactions involving H2, N2, CO, CO2 and CH4 are likely to have been central to the origin of life. This is indicated by the active-site structures of the enzymes involved, which are often reminiscent of minerals. Through the combined efforts of protein crystallography, various types of spectroscopy, theoretical calculations and model chemistry, it has been possible to put forward plausible mechanisms for gas-based metabolism by extant microorganisms. Although the reactions are based on metal centres, the protein matrix regulates reactivity and substrate and product trafficking through internal pathways, specific ligation and dielectricity.

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.

The Difference a Se Makes? Oxygen-Tolerant Hydrogen Production by the [NiFeSe]-Hydrogenase from Desulfomicrobium baculatum

Protein film voltammetry studies of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum show it to be a highly efficient H2 cycling catalyst. In the presence of 100% H2, the ratio of H2 production to H2 oxidation activity is higher than for any conventional [NiFe]-hydrogenases (lacking a selenocysteine ligand) that have been investigated to date. Although traces of O2 (<< 1%) rapidly and completely remove H2 oxidation activity, the enzyme sustains partial activity for H2 production even in the presence of 1% O2 in the atmosphere. That H2 production should be partly allowed, whereas H2 oxidation is not, is explained because the inactive product of O2 attack is reductively reactivated very rapidly, but this requires a potential that is almost as negative as the thermodynamic potential for the 2H(+)/H2 couple. The study provides further encouragement and clues regarding the feasibility of microbial/enzymatic H2 production free from restrictions of anaerobicity.

The Protein Environment Drives Selectivity for Sulfide Oxidation by an Artificial Metalloenzyme

Magic Mn–salen metallozyme: The design of an original, artificial, inorganic, complex‐protein adduct, has led to a better understanding of the synergistic effects of both partners. The exclusive formation of sulfoxides by the hybrid biocatalyst, as opposed to sulfone in the case of the free inorganic complex, highlights the modulating role of the inorganic‐complex‐binding site in the protein.

Structural characterization of a putative endogenous metal chelator in the periplasmic nickel transporter NikA

Escherichia coli and related bacteria require nickel for the synthesis of hydrogenases, enzymes involved in hydrogen oxidation and proton reduction. Nickel transport to the cytoplasm depends on five proteins, NikA-E. We have previously reported the three-dimensional structure of the soluble periplasmic nickel transporter NikA in a complex with FeEDTA(H 2O) (-). We have now determined the structure of EDTA-free NikA and have found that it binds a small organic molecule that contributes three ligands to the coordination of a transition metal ion. Unexpectedly, His416, which was far from the metal-binding site in the FeEDTA(H 2O) (-)-NikA complex, becomes the fourth observed ligand to the metal. The best match to the omit map electron density is obtained for butane-1,2,4-tricarboxylate (BTC). Our attempts to obtain a BTC-Ni-NikA complex using apo protein and commercial reagents resulted in nickel-free BTC-NikA. Overall, our results suggest that nickel transport in vivo requires a specific metallophore that may be BTC.

The Helicobacter pylori GroES Cochaperonin HspA Functions as a Specialized Nickel Chaperone and Sequestration Protein through Its Unique C-Terminal Extension

The transition metal nickel plays a central role in the human gastric pathogen Helicobacter pylori because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metals harmful effects, H. pylori is equipped with several specific nickel-binding proteins. Among these, H. pylori possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel in vitro. To investigate the function of this extension in H. pylori, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that H. pylori HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.

Cobaloxime-Based Artificial Hydrogenases

Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.

Tricarbonylmanganese(I)–lysozyme complex: a structurally characterized organometallic protein

The reaction of the new and structurally characterized covalent {Mn(CO)(3)(H(2)O)(2)}(+)-lysozyme adduct with NiS(4) and NiN(2)S(2) complexes generates binuclear Ni-Mn complexes; relevance to the reactivity of the protein-bound {Fe(CO)(CN)(2)} intermediate during maturation of [NiFe] hydrogenases is discussed.