Zeghouf M, Fontecave M, Coves J
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, CEA-Grenoble, France.
J Biol Chem. 2000 Dec 1;275(48):37651-6. doi: 10.1074/jbc.M005619200.
Escherichia coli sulfite reductase (SiR) is a large and soluble enzyme with an alpha(8)beta(4) quaternary structure. Protein alpha (or sulfite reductase flavoprotein) contains both FAD and FMN, whereas protein beta (or sulfite reductase hemoprotein (SiR-HP)) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is set up to arrange the redox cofactors in a FAD-FMN-Fe(4)S(4)-Heme sequence to make an electron pathway between NADPH and sulfite. Whereas alpha spontaneously polymerizes, we have been able to produce SiR-FP60, a monomeric but fully active truncated version of it, lacking the N-terminal part (Zeghouf, M., Fontecave, M., Macherel, D., and Covès, J. (1998) Biochemistry 37, 6114-6123). Here we report the cloning, overproduction, and characterization of the beta subunit. Pure recombinant SiR-HP behaves as a monomer in solution and is identical to the native protein in all its characteristics. Moreover, we demonstrate that the combination of SiR-FP60 and SiR-HP produces a functional 1:1 complex with tight interactions retaining about 20% of the activity of the native SiR. In addition, fully active SiR can be reconstituted by incubation of the octameric sulfite reductase flavoprotein with recombinant SiR-HP. Titration experiments and spectroscopic properties strongly suggest that the holoenzyme should be described as an alpha(8)beta(8) with equal amounts of alpha and beta subunits and that the alpha(8)beta(4) structure is probably not correct.
大肠杆菌亚硫酸盐还原酶（SiR）是一种大型可溶性酶，具有α(8)β(4)四级结构。蛋白质α（或亚硫酸盐还原酶黄素蛋白）同时含有FAD和FMN，而蛋白质β（或亚硫酸盐还原酶血色素蛋白（SiR-HP））含有与西罗血红素偶联的铁硫簇。该酶将氧化还原辅因子按FAD-FMN-Fe(4)S(4)-血红素顺序排列，形成NADPH与亚硫酸盐之间的电子传递途径。虽然α会自发聚合，但我们已经能够制备出SiR-FP60，它是α的一种单体形式但具有完全活性的截短版本，缺少N端部分（Zeghouf, M., Fontecave, M., Macherel, D., and Covès, J. (1998) Biochemistry 37, 6114 - 6123）。在此我们报道β亚基的克隆、过量表达及特性研究。纯的重组SiR-HP在溶液中表现为单体，其所有特性与天然蛋白相同。此外，我们证明SiR-FP60和SiR-HP的组合能产生一种功能性的1:1复合物，两者紧密相互作用，保留了约20%的天然SiR活性。另外，通过将八聚体亚硫酸盐还原酶黄素蛋白与重组SiR-HP一起孵育，可以重新构建出具有完全活性的SiR。滴定实验和光谱性质强烈表明，全酶应被描述为具有等量α和β亚基的α(8)β(8)结构，而α(8)β(4)结构可能不正确。
