Marta C. Marques

A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, β-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation.

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O2. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O2. The inhibition of the Ni–Fe–Se hydrogenase activity by O2 could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O2 on the bridging site between Ni and Fe.

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.

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.

Purification, crystallization and preliminary crystallographic analysis of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough.

The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] proteins in which a selenocysteine is a ligand of the Ni. These enzymes demonstrate interesting catalytic properties, showing a very high H(2)-producing activity that is sustained in the presence of low O(2) concentrations. The purification, crystallization and preliminary X-ray diffraction analysis of the [NiFeSe] hydrogenase isolated from Desulfovibrio vulgaris Hildenborough are reported. Crystals of the soluble form of this hydrogenase were obtained using 20% PEG 1500 as a precipitant and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 60.57, b = 91.05, c = 66.85 A, beta = 101.46 degrees. Using an in-house X-ray diffraction system, they were observed to diffract X-rays to 2.4 A resolution.

ATP Synthesis and Biosensing Coupled to the Electroenzymatic Activity of a Hydrogenase on an Electrode/Biomimetic Membrane Interface

Cells generate energy by coupling a proton gradient across a phospholipid bilayer membrane with the activity of a cross-membrane ATP synthase enzyme. [...]