CYTOCHROME BO FROM ESCHERICHIA-COLI - BINDING OF AZIDE TO CU-B

Azide binds to fast cytochrome bo with a stoichiometry of 1:1, the dis sociation constant for this reaction being approximately 2 x 10(-5) M. The changes induced in the electronic absorption are very slight and are consistent with heme o remaining hexacoordinate high-spin, an obse rvation confirmed by room temperature MCD spectroscopy in the region 3 50-2000 nm. X-band EPR spectroscopy of the azide-bound form shows heme o remains coupled to Cu-B, but that the integer-spin signal (g = 3.7) that we have previously reported to be associated with the binuclear center of fast cytochrome bo [Watmough et al. (1993) FEES Lett. 319, 1 51-154]. is shifted to higher field. The kinetics of azide binding are an order of magnitude faster than those observed for the binding of c yanide, Unlike cyanide, the observed rate constants do not saturate in the range 0.05-25 mM. The value of K-on shows a marked dependence on pH, indicating that the active species is hydrazoic acid. It is argued that these data are consistent with the binding of azide ion as a ter minal ligand to Cu-B yielding a binuclear center in the form Fe-III-OH 2:: Cu-B(II)-N-3. The binding of azide in heme-copper oxidases may cau se displacement of another nitrogenous ligand from Cu-B which might ex plain the absence of electron density associated with histidine-325 in the structure of the Paracoccus denitrificans CCO [Iwata et al, (1995 ) Nature 376, 660-669]. Formate appears to act as a bidentate ligand t o the binuclear center, blocking not only the binding of azide to Cue but also the binding of cyanide to heme o.