FAST CYTOCHROME BO FROM ESCHERICHIA-COLI BINDS 2 MOLECULES OF NITRIC-OXIDE AT CU-B

The reaction of nitric oxide (NO) with fast cytochrome bo from Escheri chia coli has been studied by electronic absorption, MCD, and EPR spec troscopy, Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 an d 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 spect roscopy revealed that the broad EPR signals at g approximate to 7.3, 3 .7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and Cu-B(II) (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 formati on of the 2:1 complex, which is EPR silent. The absorption spectrum sh ows that heme o remains in the high-spin Fe-III state throughout the t itration. These results are consistent with the binding of up to two N O molecules at Cu-B(II). This has been Confirmed by studies with the C l- adduct of fast cytochrome bo. MCD evidence shows that heme o remain s ligated by histidine and water. Addition of excess NO to the Cl- add uct leads to the appearance of a high-spin Fe-III heme EPR signal, Hen ce chloride ion binds to Cu-B, blocking the binding of a second NO mol ecule. These results suggest a mechanism for the reduction of NO to ni trous oxide by cytochrome bo and cytochrome c oxidase in which the bin ding of two cis NO molecules at Cu-B permits the formation of an N-N b ond and the abstraction of oxygen by the heme group.