Studies of proton pumping mechanism in BA3 type cytochrome oxidase of thermus thermophilus and CBB3 type cytochrome oxidase of vibrio cholerae

Oxygen reductase members of the heme-copper oxidoreductase superfamily are redox-driven proton pumps which couple the free energy liberated from the reduction of O2 to the translocation of protons across the membrane. The A-family requires two proton input channels (D- and K-channels) to transfer protons used for oxygen reduction chemistry and proton pumping, with the D-channel transporting all pumped protons. We use comparative genomics, site-directed mutagenesis, and redox kinetics to demonstrate that the B-family of oxygen reductases utilize only one proton input channel, structurally analogous to the K-channel, for the delivery of protons for both chemistry and pumping. Since all of the oxygen reductase families are evolutionary related and perform the same chemistry for oxygen reduction, we propose that they must also share an evolutionary conserved mechanism for proton pumping. Comparative analysis between all oxygen reductase families identifies the A-propionate of the active-site heme as the most likely proton loading and the kinetic gating site. According to the crystal structure of ba3 oxidase in T. thermophilus, two highly conserved residues, D372 and H376 which are located at the interface of subunit I and II of complex, form ion pairs with the A-propionate of active-site heme a3. A set of mutations of D372 and H376 are made and examined in this work. Interestingly, mutations of D372I and D372A still remain ~50% and 15% electron transfer activity respectively, but abolished proton translocation. Furthermore, the reduced minus oxidized FTIR difference spectra of the D372I also show significant changes in the protonated heme propionate signals. These results suggest that the H-bonding network A-propionate heme a3-D372 might play an important role in proton translocation. Heme c binding proteins have been well identified in Vibrio cholerae. Of these, two predicted c-type cytochromes, diheme c4 (encoded from gene cycA) and monoheme c5 (encodes from gene cycB), have high conservative amino acid residues in all of the Vibrio strains and are significant homologues in other known bacteria. Both of them are predicted to transfer electrons on the respiratory chain in V. cholerae. In the current work, cytochromes c4 and c5 have been cloned and expressed heterologously in E. coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e-1/s. In contrast, reduced cytochrome c5 is not a substrate for the cbb3 oxygen reductase. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 mV and 340 mV (vs SHE), similar to the electrochemical properties of other c4-type cytochromes. The majority of cytochrome c4’s are found in β- and γ- proteobacteria. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ- proteobacterial clades, supporting our proposal that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β- proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal oxidases in their respiratory chains, and the γ- proteobacterium Pseudomonas stutzeri.