Mutating the environment of heme b595 of E. coli cytochrome bd-I oxidase shifts its redox potential by 200 mV without inactivating the enzyme

Cytochrome bd-I catalyzes the reduction of oxygen to water with the aid of hemes b558, b595 and d. Here, effects of a mutation of E445, a ligand of heme b595 and of R448, hydrogen bonded to E445 are studied electrochemically in the E. coli enzyme. The equilibrium potential of the three hemes are shifted by up to 200 mV in these mutants. Strikingly the E445D and the R448N mutants show a turnover of 41 +/- 2 % and 20 +/- 4 %, respectively. Elec-trocatalytic studies confirm that the mutants react with oxygen and bind and release NO. These results point towards the ability of cytochrome bd to react even if the electron transfer is less favorable.