Direct electron transfer to cytochrome c oxidase (CcO) is investigated as a function of packing density of the surface layer. This is varied by the surface concentration of chelator molecules when the enzyme is immobilized on the electrode using the his-tag technology. Chelator molecules with a terminal nitrilotriacetic acid group are synthesized ex situ in contrast to in situ synthesis used in a previous work. Self-assembled monolayers of the chelator mixed at different mole fractions with a dilution molecule are prepared to bind the CcO after complex formation with Ni 2+ ions. The CcO, which is immobilized in the solubilized form, is then reconstituted into a proteintethered bilayer lipid membrane (ptBLM). Varying the mixing ratio of chelator to dilution molecules enabled us to control the packing density of CcO residing in the ptBLM. Subtle differences in the architecture of the protein/lipid layers revealed by surface-enhanced IR absorption spectroscopy are considered to be essential for an effective electron transfer. Cyclic voltammograms are measured under anaerobic conditions at different scan rates and analyzed by means of a model which describes the transfer of four electrons to CcO in the ptBLM. The rate constants thus obtained show a marked dependence on the packing density.

An improved setup including a measuring cell was designed for time-resolved surface-enhanced resonance Raman (SERR) spectroscopy. The cell is based on a rotating disk electrode (RDE) made from electrochemically roughened Ag. Cytochrome c (cc) adsorbed on a monolayer of mercaptoethanol is investigated with respect to heterogeneous electron transfer. Cyclic voltammograms and potential-dependent static SERR spectra indicate cc to be electroactive on the Ag electrode. The standard redox potential was found to be 234 mV. Time-resolved SERR spectra were then measured triggered by periodic potential pulses changing the protein between the oxidized and reduced state at a frequency of 10 Hz. Monoexponential functions obtained from the intensity of the band at 1361 cm-1 plotted versus time yielded the rate constants of heterogeneous electron transfer to be k(ox) = 46 +/- 7 s(-1) and k(red) = 84 +/- 20 s(-1). These relatively low rates are in line with the orientation of cc on the mercaptoethanol-modified Ag electrode. In this case the heme cleft pointed away from the surface thus hampering electron transfer.

A surface was developed for surface-enhanced resonance Raman spectroscopy in the near-ultraviolet range. For this purpose, silver nanoparticles were synthesized, exhibiting localized surface plasmon resonance at an excitation wavelength of 410 nm. The nanoparticles were adsorbed onto the plane of a silver rod where the number of particles per unit area was controlled by the adsorption time. SERR spectra of cytochrome c were used as a reference system to quantify the enhancement factor of the newly developed surface. Optimal enhancement was obtained when the individual nanoparticle coverage on the surface reached a maximum. Nanoparticles touching each other in pairs or clusters led to shifts in the resonance frequency of resonantly coupled localized surface plasmons. Clusters therefore did not contribute to the enhancement factor at the wavelength of 413 nm. The overall surface enhancement factor finally achieved at this particular wavelength was in the range of 10 4 -10 5 .