Oriented immobilization and electron transfer to the cytochrome c oxidase

Abstract

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.

Modeling direct electron transfer to a multi-redox center protein: Cytochrome c oxidase

“…Dithiobis (nitriloacetic acid butylamidyl propionate) (short: DTNTA) DTNTA was synthesized as described [18], briefly N-(5-amino-1-carboxypentyl) iminoacetic acid (ANTA) was coupled to dithiobis (N-succinimidyl propionate) (DTSP).…”

“…Immobilization of the protein was performed as described [18]. Briefly CcO from Rhodobacter sphaeroides with a his-tag engineered to the C-terminus of subunit II was expressed and purified according to Hiser et al [19].…”

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

“…Our ptBLM system has the further advantage that a thin gold layer to be used as an electrode can be placed on top of the ATR crystal of an IR spectrometer thus providing the means for coupled EC/FTIR studies. When the gold electrode is further provided with a nano-particulate surface, surface-enhanced infrared-absorption spectroscopy (SEIRAS) can be employed controlled by EC [13,[19][20][21]. This methodology had proved very useful in the past in our titration studies of the CcO as well as time-resolved (tr-) SEIRAS studies of CcO and the photosynthetic reaction center (RC) of R. sphaeroides.…”

“…SEIRAS studies in an anaerobic environment [13,[19][20][21][22] had shown that the CcO could be converted reversibly into the fully reduced state. This state is necessary as a starting point for measurements under aerobic conditions, thus enabling the enzyme to undergo enzymatic oxidation.…”

Spectro-Electrochemical Investigation of the bc1 Complex from the Yeast Saccharomyces cerevisiae using Surface Enhanced B-Band Resonance Raman Spectroscopy

“…(Friedrich, Robertson et al 2008;Nowak, Schach et al 2009;Schach, Nowak et al 2010) For this purpose the bc 1 complex from the yeast Saccharomyces cerevisiae was adsorbed on a monolayer of cysteamine (CA) self assembled on template stripped gold (TSG) and alternatively the top plane of a silver rod modified with silver nanoparticles (Fig. 1A).…”