ISSN:
1432-072X
Keywords:
b-Type cytochrome
;
Pyridine hemochromogen
;
Photoreduction
;
Cyanide
;
CO, NO binding
;
Fifth coordinate structure
;
EPR
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract A green protein from the soluble extract of anaerobically grown Bacillus halodenitrificans cells was purified and determined by non-denaturing procedures or SDS-PAGE to have a molecular mass of 64 kDa. The pyridine hemochromogen was shown to be that of a b-type cytochrome prosthetic group that was soluble in ether. The protein contained 6.2mol protoheme per mol protein-1. Photoreduction of the native protein yielded a product with an electronic absorption spectrum retaining the 559 nm maximum and the 424-nm Soret band displayed in the dithionite-reduced sample. Incubation of a reduced sample in the presence of air failed to return it to the original oxidation state. Electronic spin was not affected by pH. The reduced but not the oxidized form of the cytochrome bound cyanide, carbon monoxide, and nitric oxide, providing spectra resembling those of cytochromes c′ from several sources. Addition of nitroprusside to the reduced protein yielded a spectrum similar to that 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 nitrosylheme structure. EPR studies provided g values with the triplet spectral pattern consistent with a five-coordinate ferrous nitrosyl heme. Flushing of the NO-derivative with argon and overnight exposure to air returned the nitrosylheme to the ferric form, and EPR values confirmed the reversion. All these spectral characterizations are strikingly similar to those of soluble guanylate cyclase, including the observation 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 definitive physiological role because of its unusual properties, the green protein was named a nitric oxide-binding protein.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00263778
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