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.

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.

Photosynthetic reaction centers (RCs) from Rb. sphaeroides with a genetically engineered 7-his-tag at the C-terminus of the M-subunit are bound to a Ni-NTA-modified gold surface. Subsequently, the bound RCs are subjected to in situ dialysis in the presence of lipid micelles to form a protein-tethered lipid bilayer membrane (ptBLM). Redox properties of the RC thus immobilized are investigated by cyclic voltammetry. Photocurrrents are generated in the range of 10 μA cm −2 , however, different from previous studies at potentials of −200 and −300 mV, and without cytochrome c as a mediator. The unexpected behavior is explained in terms of an interprotein reaction between RC molecules promoted by the lipid bilayer, which we had previously detected by surface-enhanced infrared absorption spectroscopy.