Biochemical and Biophysical Research Communications, Vol.296, No.5, 1272-1278, 2002
Cytochrome bo(3) from Escherichia coli: the binding and turnover of nitric oxide
The reaction of nitric oxide (NO) with fast and reduced cytochrome b(03)(cyt bo(3)) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo(3) was determined using an NO electrode in the [NO] range 1-14 muM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo(3) corresponded to binding of 1 NO molecule per cyt bo(3) functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption (similar to0.3 mol NO mol bo(3)(-1) min(-1)), indicating that cyt bo(3) 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 muM) both fast and pulsed oxidised cyt bo(3) bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K-d = 5.6 +/- 0.6 muM 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 bo(3) 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 Cu-B(II) results in the formation of the nitrosonium (Cu-B(I)-NO+) complex. NO+ then reacts with OH-, a Cu-B 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 ho(3) does result in the reduction of Cu-B. (C) 2002 Elsevier Science (USA). All rights reserved.