University of Helsinki
Terminal oxidases are integral membrane proteins located in the inner mitochondrial membrane or in bacterial cell membrane. Cytochrome c oxidase catalyzes the final step of energy conservation in the respiratory chain. The four-electron reduction of molecular oxygen to water performed by the enzyme is coupled to proton translocation across the membrane and the created mH+ is utilized for example as the energy source in ATP synthesis. Microorganisms are often capable of expressing terminal oxidases of different types which enables them to adapt to the prevailing conditions.
Rhodobacter sphaeroides is a gram-negative facultative photosynthetic bacterium having a branched respiratory chain with two cytochrome c oxidases. In aerobic conditions the proton translocation is accomplished by cytochrome aa3 and the cbb3-type terminal oxidase is expressed under microaerobic conditions (1). Cytochrome cbb3 is encoded by a ccoNOQP gene cluster implying a four subunit composition. Subunit ccoN is predicted to form 12 to 14 membrane-spanning helices. The binuclear center, where the oxygen reduction chemistry takes place, is composed of a high-spin haem b3 and CuB. There is also a low-spin haem b present, which transfers electrons to the O2 reduction site. The subunits ccoO and ccoP have one transmembrane helix each, and there are three low-spin haems c bound, two in subunit ccoP and one in subunit ccoO. They serve as immediate electron acceptors for soluble electron donors. The role of the fourth subunit is still unknown (1,2).
Cytochrome aa3 oxidase from bacteria is homologous to the eukaryotic mitochondrial terminal oxidase. This enzyme is well characterised and the structure is resolved. Despite that the mechanism of O2 reduction and proton pumping coupled to it are still not fully understood. To gain more information about the mechanism of cytochrome c oxidases cbb3-type terminal oxidase was chosen to provide a different point of view. There is not yet a 3D structure of cytochrome cbb3, but according to the sequence alignment studies this enzyme has only a few conserved amino acids even in the region of the active site when compared to the other haem-copper oxidases.
An efficient method for expression and purification of cytochrome cbb3 has been developed providing adequate amounts of enzyme for spectroscopic, structural and functional studies. The aim of the study is to characterise the enzyme with biochemical and biophysical methods. For instance both optical and FTIR spectroscopies are used for gaining more information of the characteristics and the function of the cbb3-type cytochrome oxidase and to study proton and electron transfer in this enzyme electrometric methods are applied. Also attempts to crystallise cytochrome cbb3 oxidase are taking place.
1. García-Horsman, J.A., Berry, E., Shapleigh, J.P., Alben, J.O., Gennis, R.B. (1994) Biochemistry 33, 3113-3119.
2. Toledo-Cuevas, M., Barquera, B., Gennis, R.B., Wikström, M., García-Horsman, J.A. (1998) Biochim. Biophys. Acta 1365, 421-434.