Oriented Immobilization of a Membrane-Bound Hydrogenase onto an Electrode for Direct Electron Transfer

Abstract

The ACS journal of surfaces and colloids 27: 6449-6457 (2011) 2 ABSTRACTThe interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble than with membrane enzymes due to the higher structural complexity and instability of the latter proteins. In this work we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H 2 -oxidation reversibly and is associated to the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, only allowing mediated electron transfer between enzyme and electrode. On the other hand, a one-step immobilization procedure favours formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted in a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

“…Finally, the electrodes were incubated overnight in a 0.2 mg mL −1 proteoliposome suspension in the presence of 240 mg mL −1 Calbiosorb adsorbent biobeads (Calbiochem). Electrochemical, AFM, and QCM measurements were carried out as reported previously 16a, 17, 27. Inorganic phosphate (P i ) determination was performed by the Green Malachite assay 28.…”

“…The assembly was done sequentially, following the steps previously described to covalently bind the hydrogenase on the electrode surface with the appropriate orientation for direct electron transfer 16a, 17. Once this first protein layer was characterized, the proteoliposomes containing the E .…”

Controlling Redox Enzyme Orientation at Planar Electrodes

“…The orientation of the [NiFeSe] membrane-bound Hase from D. vulgaris Hildenborough on a gold electrode modified with SAMs and a lipid bilayer was further studied by combining AFM and electrochemistry [156]. This Hase is characterized by the presence of a lipidic tail at the opposite of the distal 4Fe4S cluster-the exit point of electrons.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…De Lacey et al proposed the controlled immobilization of a membrane-bound [NiFeSe]-Hase based on a phospholipid bilayer arranged either on top of the electrode or on top of Hase disposed on a SAM gold electrode. [203] The latter configuration was shown to help in a stable DET process for H 2 oxidation thanks to the insertion of the enzyme lipid tail in the lipid bilayer, which orients the enzyme with the distal FeS facing the electrode. The same configuration was further studied by coupling SEIRA and electrochemistry.…”