Cytochrome bo(3) from Escherichia coli: the binding and turnover of nitric oxide.
The reaction of nitric oxide (NO) with fast and reduced cytochrome bo(3)(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 microM. 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 ( approximately 0.3molNOmol 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 microM) 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 microM 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 bo(3) does result in the reduction of Cu(B).
School of Cell and Molecular Biosciences, The Medical School, University of Newcastle, NE2 4HH, Newcastle, UK. c.s.butler@ncl.ac.uk
Clive
C
Butler
Elena
E
Forte
Francesca
F
Maria Scandurra
Marzia
M
Arese
Alessandro
A
Giuffré
Colin
C
Greenwood
Paolo
P
Sarti
D001665:Binding Sites; D003300:Copper / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011485:Protein Binding
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D009573:Nitrites; C083850:cytochrome bo3, E coli; D009569:Nitric Oxide; D003300:Copper; D010088:Oxidoreductases; C024748:copper oxidase; C034747:nitric-oxide reductase