Kinetic Analysis of Inactivation and Enzyme Reaction of Oxygen-Tolerant [NiFe]-Hydrogenase at Direct Electron-Transfer Bioanode

Abstract

Membrane-bound [NiFe]-hydrogenase (MBH) from Hydrogenovibrio marinus is an O 2 -tolerant enzyme and allows direct-electron-transfer (DET) bioelectrocatalysis for H 2 -oxidation. MBH is a promising bioelectrocatalyst for bioanode of enzymatic H 2 O 2 biofuel cells. From the practical viewpoint of electricity production, the H 2 -depletion near the electrode surface and the oxidative and reversible inactivation called "anaerobic inactivation" of [NiFe]-hydrogenases limit the H 2 -oxidation at high potentials. We have already proposed a gas-diffusion system to avoid inactivation in our previous study. In this research, we have analyzed the kinetics of the electrochemically induced anaerobic inactivation and the DET bioelectrocatalytic reaction of MBH on electrodes. When the inactivation is considered as a competitive inhibition-like reaction, the maximum value of the apparent Michaelis constant reaches 6.5 mM (at Ketjen Black-modified electrode) as analyzed in our kinetic model. Since the value is larger than the saturated H 2 -concentration in solution (0.74 mM), we conclude that high-speed H 2 -supply realized by a gas-diffusion electrode is essential to compete with the inactivation. Furthermore, a gas-diffusion bioanode with MBH can eliminate the H 2 -depletion near the electrode surface and has reached about 10 mA cm ¹2 at 0 V (vs. Ag«AgCl«sat. KCl electrode) under quiescent (passive) and H 2 -atmospheric conditions.Hydrogenases are enzymes which catalyze the interconversion between hydrogen and proton.1 The two major classes of hydrogenases are known as [FeFe] or [NiFe] according to the metals in their buried active sites.1,2 Since hydrogenases are highly active, with turnover frequencies for H 2 -oxidation in excess of thousands of molecules of H 2 per second at 30°C, 3 they can be alternative catalysts instead of platinum as an H 2 O 2 fuel cell.4 From the aspect of bioelectrochemistry, hydrogenases have received a lot of attention, and the bioelectrocatalytic properties have extensively been characterized all over the world. 1,2,58 Enzymatic biofuel cells, which are electric generators that utilize redox enzymes as catalysts, can generally operate under mild conditions (neutral pH, atmospheric pressure, and room temperature). They are classified into two types: mediated electron transfer (MET)-type and direct electron transfer (DET)-type.912 MET-type cell utilizes artificial mediators to shuttle the electron between the enzyme and the electrode to reduce the kinetic hindrance in the interfacial electron transfer. On the other hand, DET-type cell does not require, in principle, any separator or membrane between the electrodes due to the mediator-less configuration. In addition, the DET-type one can reduce the possible health hazard problems caused by artificial mediators and the thermodynamic loss ascribed to the difference in the redox potentials between the mediator and the active site of the enzyme. Furthermore, the DET-type system enables us to construct a very simple and compact enzymatic biofuel ...

Bioelectrochemical analysis of thermodynamics of the catalytic cycle and kinetics of the oxidative inactivation of oxygen-tolerant [NiFe]-hydrogenase

“…As described in Introduction, we have performed kinetic analysis on the oxidative inactivation of HmMBH adsorbed on Au electrode and meso-porous carbon electrode (Ketjen Black is used as an electrode material) [41]. In this study, we performed the same analysis on HmMBH chemically immobilized on Au electrode.…”

“…HmMBHs on the Au electrode were bound with each other through amide-linkage. According to a previous study [41], we assumed a reversible (two-way) first-order reaction between the active (Ni-SI) and inactive (Ni-B) states of the enzyme with two rate constants (k I and k A ), as shown in Scheme 1, k I and k A being the apparent rate constants of inactivation and reactivation, respectively. The initial potential (E 1 ) was set at − 0.2 V, where the oxidative inactivation does not occur (or can be ignored) at pH 6 and the maximum catalytic current (i 0 ) was defined.…”

“…This reaction is called as the oxidative inactivation and is an obstacle for constructing an aforementioned bioelectrochemical device for H 2 energy conversion. Several kinetic studies have been reported [40][41][42]. In addition, there are two reports to overcome this reaction [43,44].…”

“…As far as we know, this is the first report on a description of the pseudo-steady-state current of the oxidative inhibition as a function of the electrode potential and the Michaelis constant. In the dissolved system, the saturated H 2 concentration is 740 μM at 40°C [45], which is only slightly larger than the Michaelis constant (K M = 570 μM) evaluated in DET-type bioelectrocatalysis [41]. To achieve the high H 2 concentration, we have constructed a gas-diffusion-type electrode, which can directly supply H 2 gas to the enzyme layer on the solution-side of the anode.…”

“…To achieve the high H 2 concentration, we have constructed a gas-diffusion-type electrode, which can directly supply H 2 gas to the enzyme layer on the solution-side of the anode. As a result, the high-speed (non-equilibrated but a steady-state)-H 2 -supply enhances the H 2 concentration around the hydrogenase and thus makes it possible to overcome the oxidative inactivation [41,44].…”