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

被引:13
作者
DENARIAZ, G
KETCHUM, PA
PAYNE, WJ
LIU, MY
LEGALL, J
MOURA, I
MOURA, JJ
机构
[1] UNIV GEORGIA, DEPT BIOCHEM, ATHENS, GA 30602 USA
[2] UNIV GEORGIA, DEPT MICROBIOL, ATHENS, GA 30602 USA
[3] INST TECNOL QUIM & BIOL, OEIRAS, PORTUGAL
[4] UNIV NOVA LISBOA, FAC CIENCIAS & TECHNOL, DEPT QUIM, P-2825 MONTE DE CAPARICA, PORTUGAL
关键词
B-TYPE CYTOCHROME; PYRIDINE HEMOCHROMOGEN; PHOTOREDUCTION; CYANIDE; CO; NO BINDING; 5TH COORDINATE STRUCTURE; EPR;
D O I
10.1007/s002030050144
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A green protein from the soluble extract of anaerobically grown Bacillus halodenitrificans cells was purified and determined by non-denaturin, 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 6.2 mol 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 tripler 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.
引用
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页码:316 / 322
页数:7
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