Electrochemical definitions of O2 sensitivity and oxidative inactivation in hydrogenases (original) (raw)
Related papers
Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach
The Journal of biological chemistry, 2016
[FeFe] hydrogenases catalyze rapid H 2 production but are highly O 2-sensitive. Developing O 2-tolerant enzymes is needed for sustainable H 2 production technologies, but the lack of a quantitative and predictive assay for O 2 tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O 2 sensitivity by using an assay employing ferredoxin NADP ؉ reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H 2 production in an O 2-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction solutions. The second assay validates the more convenient NADPH-driven assay, which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O 2. However, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O 2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O 2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O 2-tolerant [FeFe] hydrogenases required for photosynthetic, biological H 2 production.
Hydrogen Production under Aerobic Conditions by Membrane-Bound Hydrogenases from Ralstonia Species
Journal of the American Chemical Society, 2008
Studies have been carried out to establish the ability of O2-tolerant membrane-bound [NiFe] hydrogenases (MBH) from Ralstonia sp. to catalyze H 2 production in addition to H2 oxidation. These hydrogenases are not noted for H 2-evolution activity, and this is partly due to strong product inhibition. However, when adsorbed on a rotating disk graphite electrode the enzymes produce H 2 efficiently, provided the H 2 product is continuously removed by rapidly rotating the electrode and flowing N2 through the gastight electrochemical cell. Electrocatalytic H 2 production proceeds with minimal overpotentialsa significant observation because lowering the overpotential (the electrochemically responsive activation barrier) is seen as crucial in developing small-molecule catalysts for H 2 production. A mutant having a high KM for H2 oxidation did not prove to be a better H 2 producer relative to the wild type, thus suggesting that weak binding of H2 does not itself confer a tendency to be a H2 producer. Inhibition by H2 is much stronger than inhibition by CO and, most significantly, even O 2. Consequently, H2 can be produced sustainably in the presence of O2 as long as the H2 is removed continuously, thereby proving the feasibility for biological H2 production in air.
Journal of Electroanalytical Chemistry, 2016
Membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus (HmMBH) is an O 2-tolerant enzyme and allows direct electron transfer (DET)-type bioelectrocatalysis for the H 2 oxidation. Very fast interfacial electron transfer occurs between the [NiFe]-active site of HmMBH and the electrode, and the potential dependence of the steady-state DET-type catalytic current has been analyzed on a thermodynamic model of a two-step one-electron transfer to get a Pourbaix diagram of the catalytic center. A reversible and oxidative inactivation that occurs when the [NiFe]-hydrogenases are suffered from the oxidative stress at high electrode potentials or high solution potentials has been kinetically analyzed for the time-dependence of the steady-state catalytic current as a measure. The kinetic analysis has shown that the rate-determining step of the oxidative inactivation is not electrochemical but chemical process and that the rate of the reductive reactivation is determined by the electrochemical process. The observed catalytic waves, especially the dependence of the waves on the scan rate and the hydrogen concentration, have been well reproduced by simulation with the thermodynamic and kinetic parameters evaluated here.
Examining the possibility of expressing hydrogenase under aerobic conditions in Escherichia coli
2020
Hydrogenase is the key enabling tool for biological hydrogen production. Generally not tolerant to oxygen, hydrogenase mediated hydrogen evolution typically occur under anaerobic conditions in particular microbes fed with different carbon substrates. But, anaerobic conditions are difficult to maintain in the lab and in industry. Thus, there is interest in exploring the expression and utilization of hydrogenase enzyme under aerobic conditions with the chassis organism as <i>Escherichia coli</i>. Currently, there are two different types of hydrogenase whose biotechnological potential is under scrutiny. [FeFe] hydrogenase is not tolerant to oxygen, but, [NiFe] hydrogenase could tolerate some level of oxygen tension. Hence, [NiFe] hydrogenase offers hope for useful application in biotechnology for biohydrogen production. Encapsulated here is a short write-up on oxygen tolerance of the two main types of hydrogenase, as well as a detailed description of [NiFe] hydrogenase.
Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli
Archives of Microbiology, 2002
This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H 2 uptake in the presence of highpotential acceptors (ferricyanide and phenazine methosulfate). H 2 uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox (E h ) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E h below -80 mV. Hydrogenase 1 had maximum activity in the E h range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O 2 . Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O 2 concentrations than hydrogenase 2.
Applied and Environmental Microbiology, 2002
A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O 2 . When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O 2 , 79% N 2 ), its H 2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O 2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O 2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 M dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O 2 , potential applications of this hydrogenase are discussed.
Journal of the American Chemical Society, 2006
Studies of the catalytic properties of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans by protein film voltammetry, under a H2 atmosphere, reveal and establish a variety of interesting properties not observed or measured quantitatively with other techniques. The catalytic bias (inherent ability to oxidize hydrogen vs reduce protons) is quantified over a wide pH range: the enzyme is proficient at both H 2 oxidation (from pH > 6) and H2 production (pH < 6). Hydrogen production is inhibited by H2, but the effect is much smaller than observed for [NiFe]-hydrogenases from Allochromatium vinosum or Desulfovibrio fructosovorans. Under anaerobic conditions and positive potentials, the [FeFe]-hydrogenase is oxidized to an inactive form, inert toward reaction with CO and O2, that rapidly reactivates upon one-electron reduction under 1 bar of H2. The potential dependence of this interconversion shows that the oxidized inactive form exists in two pH-interconvertible states with pKox ) 5.9. Studies of the CO-inhibited enzyme under H2 reveals a strong enhancement of the rate of activation by white light at -109 mV (monitoring H2 oxidation) that is absent at low potential (-540 mV, monitoring H + reduction), thus demonstrating photolability that is dependent upon the oxidation state.