Cytochrome c and Cytochrome c Oxidase:  Monolayer Assemblies and Catalysis

Abstract

A novel electrochemical system has been designed and assembled to study the kinetic activity of cytochrome c oxidase. Gold electrodes coated with 3-mercapto-1-propanol formed the surface for the physisorption of monolayers of cytochrome c and cytochrome c oxidase or a preformed cytochrome c−cytochrome c oxidase complex. The films were investigated by cyclic voltammetry at scanning at rates slow enough to permit near redox equilibrium between electrode and redox protein and hence obtain redox midpoint potentials. Cytochrome c monolayers alone displayed a reversible midpoint potential at pH 8 (E m8 vs NHE) at +240 mV, close to the native cytochrome c value observed in solution. In contrast, oxidase monolayers alone failed to support any detectable redox contact between electrode and protein, implying that the distances between the oxidase redox cofactors in the adsorbed oxidase are too far away from the electrode to promote significant electron transfer rates. However, adsorption of a preformed cytochrome c−cytochrome c oxidase complex promoted effective redox contact, demonstrating electron transfer with an apparent onset halfpoint potential at +225 mV. This effect is consistent with the mandatory requirement for cytochrome c to mediate electrons from the electrode to cytochrome c oxidase and presumably in a way reflecting the physiological pathway. Cyclic voltammetric measurements arranged to determine the rates of electron transfer between electrode and the complex showed that at scan rates up to 50 mV/s, extraordinary kinetic turnover is displayed attributable to the catalysis of oxygen reduction. Thus it is established that the protein complex can be assembled and enable the natural mediation of electron transfer from the electrode by cytochrome c to the enzyme at a rate fast enough for catalysis to be observed and controlled.

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

“…This value is very close to that determined by Haas et al (225 mV vs NHE). 27 In the absence of the electron donor cyt c (lower trace in Figure 9), CcO exhibits only a capacitive current of the dielectric layer, but the current indicating electrocatalytic activity is not observed. This suggests that CcO is not electrically coupled to the electrode, and thus direct electron transfer does not take place.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…[331,332] Moreover, many of the terminal oxidases currently reported reduce oxygen at a much lower potential than MCOs. [333] Some bacterial metabolisms in the biodiversity pool are known to involve high-potential electron-transfer chains. [334,335] Identification of high redox potential CcOs would be an original way to address the issue of low oxygen availability in H 2 /O 2 EBFCs.…”

Emprego de monocamadas auto-organizadas no desenvolvimento de sensores eletroquímicos

“…Um emparelhamento perfeito entre os alcanotióis (geralmente com uma inclinação de 20 a 30 graus em relação à normal da superfície) e o retículo do ouro resulta numa estrutura altamente ordenada (Figura 2) e tem feito deste sistema objeto de um grande número de estudos por meios espectroscópicos, microscópicos e eletroquímicos 12 . Vári-os trabalhos têm sido dedicados ao entendimento da estrutura e do ordenamento das monocamadas, que são caracterizadas pelo alto grau de organização e têm sido empregadas em estudos de transferência de elétrons [34][35][36][37][38][39] , adsorção de proteínas 38,[40][41][42][43] e, principalmente, em vários campos da eletroanalítica 12,[19][20][21][22][44][45][46][47][48][49][50][51][52][53] . A preparação da monocamada possui um papel fundamental no desempenho eletroanalítico do eletrodo modificado.…”