Simulating the slow to fast switch in cytochrome c oxidase catalysis by introducing a loop flip near the enzyme's cytochrome c (substrate) binding site.

The mitochondrial enzyme cytochrome c oxidase catalyzes the reduction of molecular oxygen in the critical step of oxidative phosphorylation that links the oxidation of food consumed to ATP production in cells. The enzyme catalyzes the reduction of oxygen at two vastly different rates that are thought to be linked to two different conformations but the conformation of the "fast enzyme" remains obscure. In this study, we demonstrated how oxygen binding at haem a3 could trigger long-distance conformational changes and then simulated a conformational change in an eight-residue loop near the enzyme's substrate (cytochrome c) binding site. We then used this modified cytochrome c oxidase (COX) to simulate a stable COX-cytochrome c enzyme-substrate (ES) complex. Compared to ES complexes formed in the absence of the conformation change, the distance between the redox centers of the two proteins was reduced by half and instead of nine, only four COX amino acid residues were found along the axis linking the electron entry point and the CuA redox center of COX: We proposed that intramolecular electron transfer in COX occurs via a charge/hydrogen relay system involving these four residues. We suggest that the conformational change and resulting shortened electron pathway are features of fast-acting COX.

Redox equilibration after one-electron reduction of cytochrome c oxidase: radical formation and a possible hydrogen relay mechanism.

Kinetic studies using UV/visible and EPR spectroscopy were carried out to follow the distribution of electrons within beef heart cytochrome c oxidase (CcO), both active and cyanide-inhibited, following addition of reduced cytochrome c as electron donor. In the initial one-electron reduced state the electron is shared between three redox centers, heme a, CuA and a third site, probably CuB. Using a rapid freeze system and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) a protein radical was also detected. The EPR spectrum of the DMPO adduct of this radical was consistent with tyrosyl radical capture. This may be a feature of a charge relay mechanism involved in some part of the CcO electron transfer system from bound cytochrome c via CuA and heme a to the a3CuB binuclear center.

Substrate binding-dissociation and intermolecular electron transfer in cytochrome c oxidase are driven by energy-dependent conformational changes in the enzyme and substrate.

Reduction of O2 by cytochrome c oxidase (COX) is critical to the cellular production of adenosine-5'-triphosphate; COX obtains the four electrons required for this process from ferrocytochrome c. The COX-cytochrome c enzyme-substrate complex is stabilized by electrostatic interactions via carboxylates on COX and lysines on cytochrome c. Conformational changes are believed to play a role in ferrocytochrome c oxidation and release and in rapid intramolecular transfer of electrons within COX, but the details are unclear. To gather specific information about the extent and relevance of conformational changes, we performed bioinformatics studies using the published structures of both proteins. For both proteins, we studied the surface accessibility and energy, as a function of the proteins' oxidation state. The residues of reduced cytochrome c showed greater surface accessibility and were at a higher energy than those of the oxidized cytochrome c. Also, most residues of the core subunits (I, II, and III) of COX showed low accessibility, ∼35%, and compared to the oxidized subunits, the reduced subunits had higher energies. We concluded that substrate binding and dissociation is modulated by specific redox-dependent conformational changes. We further conclude that high energy and structural relaxation of reduced cytochrome c and core COX subunits drive their rapid electron transfer.

Serum cytochrome c detection using a cytochrome c oxidase biosensor.

Within the last ten years it has emerged that the release of cytochrome c plays a critical role in the important process of programmed cell death. It has also been shown that this protein is released into the circulating blood following MIs (myocardial infarctions). Methods for the detection of this protein have therefore become important. The enzyme cytochrome c oxidase is specific for cytochrome c. Bovine cytochrome c oxidase was successfully immobilized in a didodecyldimethylammonium bromide vesicular system on to gold electrodes and its interaction with cytochrome c was studied. Square-wave-voltammetric analysis of the biosensor showed two redox couples with midpoint potential, E(0)', values of +182 and +414 mV compared with Ag/AgCl. The redox couple with E(0)' of +414 mV showed a cathodic sensitivity to the presence of cytochrome c in both buffer solution and human serum. Responses of the cytochrome c oxidase biosensor to oxidized cytochrome c followed hyperbolic electrochemical Michaelis-Menten kinetics with a K(m) of 1.57 microM and maximum current (I(max)) of 1.38 x 10(-6) muA. The detection limit of the biosensor in human serum was 0.2 microM, which is well below the lowest physiological concentration of 0.8 muM previously reported for MIs [Alleyne, Joseph and Sampson (2001) Appl. Biochem. Biotechnol. 90, 97-105]. These results indicate that the cytochrome c oxidase biosensors could be used to determine variations in cytochrome c concentration and thus have potential to be used as a diagnostic tool in the detection of MIs and possibly also in the study of programmed cell death.