Direct electron transfer to Cytochrome c Oxidase (CcO) was investigated using fast scan cyclic voltamme try The enzyme was tethered to the electrode in a strict orientation by means of a histidine tag with Cu-A the first electron acceptor directed towards the electrode A lipid bilayer was then reconstituted in situ around the bound proteins forming a protein tethered bilayer lipid membrane Cyclic voltammograms were measured under anaerobic conditions at different scan rates with CcO in the non-activated and the activated state The activated state was attained after catalytic turnover of the enzyme in the presence of oxygen A four-electron transfer model was developed to analyze the data This enables us to discriminate between the mechanisms of sequential and Independent electron transfer to the four redox centers Cu-A heme a heme a(3) and Cu-B Moreover values of parameters such as standard redox potentials and kinetic coefficients of electron transfer could be obtained Based on these results we conclude that direct electron transfer to CcO most likely follows the sequential mechanism thus mimicking the electron transfer form cytochrome c the genuine electron donor of CcO (C) 2010 Elsevier B V All rights reserved