AN UNUSUAL HEMOPROTEIN CAPABLE OF REVERSIBLE BINDING OF NITRIC-OXIDE FROM THE GRAM-POSITIVE BACILLUS HALODENITRIFICANS

A green protein from the soluble extract of anaerobically grown Bacill us halodenitrificans cells was purified and determined by non-denaturi n, procedures or SDS-PAGE to have a molecular mass of 64 kDa. The pyri dine hemochromogen was shown to be that of a b-type cytochrome prosthe tic group that was soluble in ether. The protein contained 6.2 mol pro toheme per mol protein(-1). Photoreduction of the native protein yield ed a product with an electronic absorption spectrum retaining the 559- nm maximum and the 424-nm Soret band displayed in the dithionite-reduc ed sample. Incubation of a reduced sample in the presence of air faile d to return it to the original oxidation state. Electronic spin was no t affected by pH. The reduced but not the oxidized form of the cytochr ome bound cyanide, carbon monoxide, and nitric oxide, providing spectr a resembling those of cytochromes c' from several sources. Addition of nitroprusside to the reduced protein yielded a spectrum similar to th at of the NO reacted protein. Nitric oxide failed to reduce the green protein. The position of the Soret band in the spectrum of the nitric oxide derivative of the green protein suggested a fifth-coordinate nit rosylheme structure. EPR studies provided g values with the tripler sp ectral pattern consistent with a five-coordinate ferrous nitrosyl heme . Flushing of the NO-derivative with argon and overnight exposure to a ir returned the nitrosylheme to the ferric form, and EPR values confir med the reversion. All these spectral characterizations are strikingly similar to those of soluble guanylate cyclase, including the observat ion that NO was reversibly bound to the protein. EPR spectra of whole cells also displayed the hyperfine lines typical of a nitrosyl-ferrous heme, accentuated when dithionite was added. In the absence of a defi nitive physiological role because of its unusual properties, the green protein was named a nitric oxide-binding protein.