The presence of a SO molecule in [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki as detected by mass spectrometry

被引:19
作者
Higuchi, Y [1 ]
Toujou, F
Tsukamoto, K
Yagi, T
机构
[1] Kyoto Univ, Grad Sch Sci, Div Chem, Sakyo Ku, Kyoto 6068502, Japan
[2] Matsushita Technores Inc, Characterizat Technol Grp, Moriguchi, Osaka 5700005, Japan
[3] Shizuoka Univ, Shizuoka 4228529, Japan
关键词
hydrogenase; Ni-Fe active site; sulfur oxide; mass spectrometry; Fourier transform infrared spectrometry;
D O I
10.1016/S0162-0134(00)00081-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The active site of [NiFe] hydrogenase is a binuclear metal complex composed of Fe and Ni atoms and is called the Ni-Fe site, where the Fe atom is known to be coordinated to three diatomic ligands. Two mass spectrometric techniques, pyrolysis-MS (pyrolysis-mass spectrometry) and TOF-SLMS (time-of-flight secondary ion mass spectrometry), were applied to several proteins, including native and denatured forms of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F, [Fe4S4](2)-ferredoxin from Clostridium pasteurianum, [Fe2S2]-ferredoxin from Spirulina platensis, and porcine pepsin. Pyrolysis-MS revealed that only native hydrogenase liberated SO/SO2 (ions of m/z 48 and 64 at an equilibrium ratio of SO and SO2) at relatively low temperatures before the covalent bonds in the polypeptide moiety started to decompose. TOF-SIMS indicated that native Miyazaki hydrogenase released SO/SO2 (m/z 47.97 and 63.96) as secondary ions when irradiated with a high-energy Ga+ beam. Denatured hydrogenase, clostridial ferredoxin, and pepsin did not release SO as a secondary ion. The FT-IR spectrum of the enzyme suggested the presence of CO and CN. These lines of evidence suggest that the three diatomic ligands coordinated to the Fe atom at the Ni-Fe site in Miyazaki hydrogenase are SO, CO, and CN. The role of the SO ligand in helping to cleave H-2 molecules at the active site and stabilizing the Fe atom in the diamagnetic Fe(II) state in the redox cycle of this enzyme is discussed. (C) 2000 Elsevier Science S.A. All rights reserved.
引用
收藏
页码:205 / 211
页数:7
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