Time-Resolved Surface-Enhanced IR-Absorption Spectroscopy of Direct Electron Transfer to Cytochrome c Oxidase from R. sphaeroides

Abstract

Time-resolved surface-enhanced IR-absorption spectroscopy triggered by electrochemical modulation has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. Single bands isolated from a broad band in the amide I region using phase-sensitive detection were attributed to different redox centers. Their absorbances changing on the millisecond timescale could be fitted to a model based on protonation-dependent chemical reaction kinetics established previously. Substantial conformational changes of secondary structures coupled to redox transitions were revealed.

Application of Polarization Modulation Infrared Reflection Absorption Spectrosocpy Under Electrochemical Control for Structural Studies of Biomimetic Assemblies

“…Moreover, both macroscopic and microscopic properties of a biological membrane such as permeability to ions, capacitance, lateral composition, concentration, orientation and physical state of lipid molecules and the activity and structure of associated proteins can be affected by drugs, changes in electrolyte composition and concentration or the static electric field [144,145]. A combination of IRS, sensitive to the structure of lipid and proteins molecules, to electrochemistry testing macroscopic properties of model membranes such as redox activity, membrane capacity or permeability to ions, giving a possibility to monitor simultaneously potential-dependent changes in lipid bilayers arising from the lipid-protein interaction [39][40][41][42]146] or redox activity of a protein embedded in the membrane [147][148][149][150]. Despite the fact that spectroelectrochemistry provides essential information concerning the structure of molecules at the sub-molecular level, the structure of the entire system exposed to physiological electric fields, these studies are rather rare.…”

“…Despite the fact that spectroelectrochemistry provides essential information concerning the structure of molecules at the sub-molecular level, the structure of the entire system exposed to physiological electric fields, these studies are rather rare. Predominantly, they concern surface enhanced infrared absorption spectroscopic studies under electrochemical control of transmembrane proteins, involved in redox-reactions, embedded into models of lipid membranes [24,[147][148][149][150]. Below results reporting the application of PM IRRAS under electrochemical control for studies of structural changes in lipid bilayers experiencing specific interactions with proteins are summarized [39][40][41][42].…”

Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS

“…2B) similar to the ones obtained under anaerobic conditions. Differences were revealed, however, when the spectra were subjected to the socalled phase sensitive detection (PSD), described in [23] and in more detail in [22]. Although changes were noticeable also in the fingerprint region, we focused on the much more pronounced amide I region.…”

“…Subsequently, SEIRA measurements in the step-scan mode were conducted, under the same conditions, e.g. at a modulation frequency of 100 Hz used in a study of Schwaighofer et al [23], with the only exception of varying the potential from -800 mV to OCP. Oxygen concentration was kept constant at 0.02 mg/l (1.25µM), although the standardization of the oxygen concentration proved difficult.…”

“…In this elevated state, the enzyme can be expected to undergo reversble conformational changes of secondary structures as a consequence of ET different from the ones before the transformation. This assumption will be tested in this study comparing our previous tr-SEIRAS studies of the CcO conducted under anaerobic conditions [22,23] with those carried out under aerobic conditions.…”