Purification and Characterization of Cytochrome C Oxidase from the Extremely Thermophilic Aerobic Bacterium, Thermus Thermophilus.

Abstract

This thesis describes purification procedures for several respiratory proteins from Thermus thermophilus, and gives accounts of their physical, chemical and functional characterizations. The rationale for this work is predicated on the following conceptions: (a) that proteins from thermophilic organisms will be generally more stable than their mesophilic counterparts; (b) that basic functional and structural similarities are found between respiratory proteins of thermophilic bacteria and mitochondria; (c) that use of bacterial enzymes would allow opportunities for experiments requiring isotopic substitutions and /or genetic manipulations. Large scale purification procedures were developed for cytochrome c oxidase, cytochrome c(,552) and cytochrome c(,555,549) from bacteria grown in a chemically defined culture medium. Detailed investigations of the cytochrome c oxidase established the following points: (1) The oxidase is of the aa(,3)-type and is strongly associated with a c-type cytochrome having properties similar to the mitochondrial cytochrome c(,1). (2) The purified enzyme is composed of two subunits in a 1 : 1 ratio, having molecular weights of 33,000 and 55,000; heme C is bound to the smaller subunit. (3) The ascorbate oxidase activity of the enzyme exhibited simple Michaelis-Menten kinetic behavior when an artificial dye, TMPD {N,N,N',N'-tetramethyl-p-phenylenediamine} or cytochromes c from various organisms, including T. thermophilus, were used as mediators. (4) An uncoupler sensitive proton pumping activity was demonstrated by observing proton extrusion from lipid vesicles reconstituted with the oxidase. The overall results strongly suggest that T. thermophilus enzyme is a single subunit aa(,3)-type oxidase which possesses active sites identical to the more complex eukaryotic enzyme and functions in a generally identical manner.