Bioenergetics at the gold surface: SEIRAS probes photosynthetic and respiratory reactions at the monolayer level

The present study surveys a novel approach to studies of membrane proteins whose catalytic action is driven by the redox potential or by the membrane potential. We introduce SEIRAS (surface-enhanced IR absorption spectroscopy) to probe a monolayer of membrane protein adhered to the surface of a gold electrode. SEIRAS renders high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude. it is demonstrated that reaction-induced spectroscopy is applicable by recording IR differences of cytochrome c after stimulation by the electrical potential. The impact of the membrane potential on the function of a membrane protein is demonstrated by performing light-induced difference spectroscopy on a microbial rhodopsin (sensory rhodopsin II) under voltage-clamp conditions. The methodology presented opens new avenues to study the mechanism of electron-triggered and voltage-gated proteins at the level of single bonds. As many of these catalytic reactions are of vectorial nature, control on the orientation of the membrane protein is mandatory. Approaches are presented on how to specifically adhere photosynthetic and respiratory proteins to the electrode surface and reconstitute these membrane proteins in the lipid bilayer. Functionality of such biomimetic systems is assessed in situ by spectro-electrochemical methods.