Studies of respiratory proteins from the thermophilic aerobic bacterium, Thermus thermophilus, and purification of cytochrome ba3, a new terminal oxidase.

Abstract

This thesis describes (1) the purification and initial characterization of cytochromes $ba\sb3$ and $b\sb{562}$, two new respiratory proteins from plasma membranes of the thermophilic, aerobic bacterium, Thermus thermophilus, and (2) studies of the reaction of cytochrome $c\sb{1}aa\sb3$, a Thermus terminal oxidase with peroxides. Cytochromes $ba\sb3$ catalyzes the consumption of O$\sb2$ in the presence of the substrates, horse heart cytochrome c, and the artificial dye TMPD. Metal analysis and pyridine hemochrome spectra revealed the presence of heme A, heme B, and Cu in a ratio of $\sim$1:1:2. Amino acid analysis yielded a minimum molecular weight of 34,500, including $\sim$1 cysteine and $\sim$9 hisitidines, per heme A. EPR spectra showed (1) resonances attributed to low-spin heme, and (2) features similar to those exhibited by Cu$\sb{\rm A}$ of $aa\sb3$-type oxidases; only 50% of the total Cu and 50% of the total Fe were detectable. Mossbauer spectra of the oxidized protein showed a component due to a paramagnetic, low-spin cytochrome, and components due to a coupled cytochrome. Our preliminary interpretation is that this protein contains heme A and one Cu ion coupled in a manner similar to cytochrome $a\sb3$ and Cu$\sb{\rm B}$ of $aa\sb3$-type oxidases, and two magnetically isolated chromophores: heme B and a second Cu ion. Metal, protein, and amino acid analyses, pyridine hemochrome spectra, and denaturing gel electrophoresis of cytochrome $b\sb{562}$ were consistent with $\sim$2 hemes B per $\sim$31,000 kDa. Reduced cytochrome $b\sb{562}$ readily transferred electrons to cytochrome c, and thus appeared to have a relatively low reduction potential ($<$+ 250 mV). Cytochrome $b\sb{562}$ had no oxidase activity, and does not belong to the class of heme B-containing terminal oxidases known as cytochromes o. The function of cytochrome $b\sb{562}$ is presently unknown; however, EPR spectra showed a resonance at g $\sim$ 3.6 similar to resonances exhibited by the hemes B of the mitochondrial $bc\sb1$ complex. Peroxo-complexes formed by oxidized cytochrome $c\sb{1}aa\sb3$ with different organic hydroperoxides were spectrally similar. Addition of reducing agents or CO to solutions of the peroxo-complex under anaerobic conditions regenerated oxidized enzyme. Mossbauer studies of the peroxo-complex revealed that 70% of the enzyme reacted with peroxide and exhibited parameters consistent with a mixture of two different components, each having a valence of Fe(IV) at the cytochrome $a\sb3$ site. Possible structures for the peroxo-complex are discussed.