The Purification and Characterization of cbb3 from Rhodobacter sphaeroides

Cytochrome c oxidase (CcO), the terminal enzyme in the respiratory electron transport chain of eukaryotes and many prokaryotes, plays a key role in energy production of aerobic organisms. The enzyme catalyzes the reduction of dioxygen to water, which is coupled to the translocation of protons across the inner mitochondrial or bacterial plasma membrane to generate ATP. The cbb₃ oxidases are the only heme-copper oxidases present in some human pathogens, including Helicobacter pylori (a major cause of gastric ulcers) and Neisseria meningitides, and thus are potential drug targets. A series of techniques, including French press, centrifugations and a Ni-based affinity column was used to grow the Rhodobacter sphaeroides cbb₃ bacterial strain and extract and purify the cbb₃ oxidase. The purity of the enzyme was investigated by its absorption spectra and by monitoring the carbon monoxide (CO) rebinding to cbb₃ following CO flash-photolysis of the fully reduced CO-bound enzyme. We predict that CO binds both to cytochromes c and b₃. Singular value decomposition (SVD) and global exponential fitting using the MATLAB program will be used to analyze the data and extract the spectra of the respective intermediates. The observed rates of CO rebinding to heme c and heme b₃ will be compared to those in the literature.