Electrochemistry, AFM, and PM‐IRRA Spectroscopy of Immobilized Hydrogenase: Role of a Hydrophobic Helix in Enzyme Orientation for Efficient H₂ Oxidation

Abstract

Nickel-iron hydrogenase ([NiFe] Hase) catalyzes hydrogen splitting into protons and electrons, and is a potential biocatalyst in fuel cells.[1] Three FeS clusters aligned as a conductive wire drive electrons from the [NiFe] active site to the surface of the enzyme, where the redox partner (including the electrode) binds. Direct enzyme connection gave access to thermodynamic and kinetic data of enzymatic reactions through direct electron transfer (DET). Mediated electron transfer (MET) allowed recreation of the physiological electron-transfer chain, and/or connection of unfavorably oriented enzymes. [2][3][4][5][6] Previous work demonstrated that DET or MET processes for H 2 oxidation by a soluble, O 2 -sensitive [NiFe] Hase from Desulfovibrio species could be controlled by electrostatic interaction. [7] The presence of an acidic patch of amino acids, coupled to a dipole moment pointing towards the distal FeS cluster (positioned at the surface of the enzyme), allowed orientation of the enzyme, which turned upside down as a function of the charge on the electrochemical interface.Recently, we reported on the electrochemistry of membrane-bound Aquifex aeolicus (Aa) [NiFe] Hase, which exhibits outstanding resistance to O 2 , CO, and heat. [8][9][10] Efficient immobilization of this Hase was achieved on graphite electrodes, in aqueous electrolytes and ionic liquids, by encapsulation in carbon nanotube networks, or connection to a redox polymer. [11,12] In contrast to the soluble, O 2 -sensitive [NiFe] Hase, no specific orientation could be obtained by electrostatic interaction for Aa Hase, and thus control of the electron-transfer process was not possible. A model structure accordingly put forward a very different environment of the distal FeS cluster, with no charged amino acid patch, in accordance with the membrane anchorage. [12] We analyze herein H 2 oxidation by Aa Hase immobilized on self-assembled monolayers (SAMs) on gold electrodes as a function of both the length and the nature of the thiol derivative (see SI 1 and SI 2 in the Supporting Information). For the first time, AFM and polarization modulation infrared reflection adsorption (PM-IRRA) studies are reported for understanding Aa Hase orientation and its consequences for electron-transfer process in H 2 oxidation.Positively charged 4-aminothiophenol (ArNH 2 ) and negatively charged 6-mercaptohexanoic acid (C 5 COOH) SAMs both yield DET and MET processes for H 2 oxidation (Figure 1 a and b), and neither process is favored over the other. A mixed process was similarly observed for H 2 oxidation at charged short-chain alkanethiols, which are known to be more disordered.[8] This strongly suggests that electroenzymatic H 2 oxidation is linked to multiple orientations of Hase on top of the charged SAMs, and not to Hase present inside possible SAM defects. The lipophilic methylene blue (MB) molecule

Controlling Redox Enzyme Orientation at Planar Electrodes

“…In another example, A. aeolicus membrane-bound Hase immobilized on hydrophobic SAM was examined with AFM. Once again, approximately 3 nm high spherical features were observed, thus indicating the presence of a monolayer of enzyme but not which orientation was adopted [142]. A similar result was obtained with another globular enzyme-R. eutropha membrane-bound Hase-immobilized on a gold electrode [149].…”

“…This strategy was applied to various enzymes immobilized on thiol-based SAMs and allowed to determine the surface chemistry on the electrode able to promote DET. One can cite relevant studies that analyzed the catalytic efficiency of A. aeolicus [NiFe]-Hase [142] on negative, positive, or hydrophobic SAMs on gold, of D. gigas Hase on positive SAM on gold [143], of R. eutropha [NiFe]-Hase on negative SAM on silver [61], of Trametes hirsuta LAC on neutral hydrophobic SAM on gold [144], of Cerrena unicolor C139 LAC positive SAM on gold [107], or of Myrothecium verrucaria BOD on positive or negative SAMs on gold [145] (Figure 7). In the latter paper, electrochemistry was coupled to SPR demonstrating that the switching between DET over MET when adsorption was performed either on negative or positive SAM was effectively linked to a different enzyme orientation and not to different enzyme loading.…”

“…Polarization Modulated Infrared Reflection Absorption Spectroscopy (PM-IRRAS) allowed the observation of different orientations of the membrane-bound Hase of A. aeolicus [142]. Indeed, depending on the physico-chemical properties of the SAM-modified gold electrode (hydrophilic or hydrophobic), the intensity of the ratio of amide I to amide II varied, reflecting different orientations of the enzyme.…”

Exploring Properties of a Hyperthermophilic Membrane‐Bound Hydrogenase at Carbon Nanotube Modified Electrodes for a Powerful H2/O2 Biofuel Cell

“…We earlier demonstrated that the orientation of immobilized MbH1 cannot be controlled simply by electrostatic interaction because of the absence of a high dipole moment and a charged patch surrounding the distal FeS cluster situated at the surface of the enzyme [28]. In addition, we have shown that a hydrophilic domain near the distal FeS cluster is formed by the trans-membrane helix surrounded by neutral detergent.…”

“…This hydrogenase from Aa (MbH1) is involved in a complex with a diheme cytochrome b anchored in the membrane, coupling H 2 oxidation to O 2 reduction. Efficient MbH1 immobilization for direct H 2 oxidation was achieved by different strategies like (i) adsorption on pyrolytic graphite electrode (PG) [26], in buffer electrolytes and room temperature ionic liquids [27], (ii) adsorption on self-assembled-monolayer (SAM) modified gold electrode [28], or (iii) connection to a redox polymer film [29]. As MbH1 is membranebound, encapsulation in liposomes was shown to enhance the enzyme stability at the electrode [30].…”