Cytochrome bo3 from Escherichia coli: The binding and turnover of nitric oxide

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo3(cyt bo3) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo3 was determined using an NO electrode in the [NO] range 1-14 ?M. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo3 corresponded to binding of 1 NO molecule per cyt bo3 functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption (~0.3 mol NO mol bo3 -1 min-1), indicating that cyt bo3 possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1-14 ?M) both fast and pulsed oxidised cyt bo3 bind NO with a stoichiometry of 1:1 with an observed dissociation constant of Kd = 5.6 ± 0.6 ?M and that NO binding was inhibited by the presence of Cl-. The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo3. These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to CuB II results in the formation of the nitrosonium (CuB I-NO+) complex. NO+ then reacts with OH-, a CuB ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo3 does result in the reduction of CuB. © 2002 Elsevier Science (USA). All rights reserved.