Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB.
The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.
Butler
C S
CS
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
Seward
H E
HE
Greenwood
C
C
Thomson
A J
AJ
D001426:Bacterial Proteins / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006418:Heme / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry
D001426:Bacterial Proteins; D002712:Chlorides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D009569:Nitric Oxide; D006418:Heme; D003300:Copper; C017168:cytochrome bo, E coli