Cytochrome c oxidase (Cox) accepts electrons from its substrate, cytochrome c and passes these to oxygen, which is reduced to water. Kinetic studies show that an active form of the enzyme (pulsed) and a slower form (resting) exists. More efficient internal electron transfer and the switching of the enzyme's oxygen/ligand binding site between opened and closed positions are said to account for the different rates of reduction. We employed bio-computing to analyse the structure of the oxygen/ligand binding site of bovine Cox under different redox states; a comparison with Thermus thermophilus Cox was also conducted. The study detected that the ligand binding site of Cox is exposed to the contents of the intermembrane space, and that the side chain of haem a 3 , located at the enzyme's oxygen/ligand binding site, approached Pro-69 and Ile-34 in faraway subunit-II. However, no open-to-closed gating structures were detected at the ligand binding site. We concluded that the resting-to-pulse transition in Cox does not involve opening-up of the ligand binding site. We propose that the rates of ligand/oxygen/cyanide binding are partly controlled by "queuing" near the binding site and that the binding of oxygen to haem a 3-CuB triggers the resting-to-pulsed transition via long-range conformational changes.
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.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.