Heme-copper oxidases and nitric oxide : Reaction mechanisms and supercomplex formation

Sammanfattning: In the denitrification process where nitrate is stepwise reduced to nitrogen gas, the toxic molecule nitric oxide (NO) is formed as an intermediate. Nitric oxide is produced by the enzyme cd1 Nitrite reductase (cd1NiR) by reduction of nitrite and is later reduced to nitrous oxide by nitric oxide reductase (cNOR).We have investigated if a complex, with the role to facilitate rapid removal of NO, is formed between the two enzymes cd1NiR and cNOR in the bacterium P. denitrificans. Using activity measurements, we found transient interactions between the two enzymes influencing enzymatic activity of cNOR and dimerization of cd1NiR. This complex formation may be important in the transition between aerobic and anaerobic respiration of the bacteria.Heme-copper oxidases are terminal components of the respiratory chain, catalysing the reduction of oxygen to water. We have investigated possible supercomplex formation between the bc1-complex and the heme-copper oxidase of either the aa3 or cbb3 type, in the respiratory chain of Rhodobacter sphaeroides. We found evidence for a functional supercomplex between bc1-aa3, but not between bc1-cbb3.The C-type (cbb3) oxidase from Vibrio cholerae has one proton transfer pathway for delivering protons both to the catalytic site and for protons being pumped. We identified an internal proton donor (XH), which could act as a branching point in the pathway. We also suggest which residues may function as this branching point; Y321 or Y321 and N293 together with water molecules. We were also interested in the C-type oxidase from Helicobacter pylori and its interaction with NO. We successfully expressed and purified an active H. pylori cbb3 in V. cholerae. We found that this cbb3 was reversibly inhibited by nitric oxide similar to other oxidases, and that it also displayed a nitric oxide reductase activity.