PURIFICATION AND PARTIAL CHARACTERIZATION OF 2 CYTOCHROME OXIDASES (CAA3 AND O) FROM THE THERMOPHILIC BACTERIUM PS3

Two cytochrome oxidases, cytochrome aa3 (EC 1.9.3.1) and cytochrome o, were purified from the membranes of a thermophilic bacterium, PS3. The enzymes were solubilized with Triton X-100 and purified to apparent homogeneity on anion-exchange columns. The properties of the 3-subunit cytochrome oxidase complex caa3 obtained here are compared with the same enzyme isolated by Sone and Yanagita. On storage, the purified caa3 enzyme undergoes denaturation; a shoulder at 432 nm seen in (CO-reduced)-minus-reduced difference spectra may be due in part to denaturation products of the enzyme. The purified cytochrome o is more stable. At room temperature, the reduced-minus-oxidized difference spectrum shows absorbance maxima at 427 and 559 nm; at 77.degree. K, its .alpha.-band is split into 554 and 557 nm components. At room temperature, the CO-reduced-minus-reduced spectrum shows troughs at 430 nm and 560 nm. Dissociating polyacrylamide gel electrophoresis suggests that the purified cytochrome o is composed of 1 type of subunit with an apparent MW of 47,000-48,000. Metal analysis of the purified enzyme demonstrated the lack of Cu. Both oxidases, purified in the presence of Triton X-100, exist in highly polydisperse forms.