Gruez A, Pignol D, Zeghouf M, Covès J, Fontecave M, Ferrer J L, Fontecilla-Camps J C
Laboratoire de Cristallographie et Cristallogenèse des Protéines Institut de Biologie Structurale J.P. Ebel, CEA-CNRS, Grenoble, France.
J Mol Biol. 2000 May 26;299(1):199-212. doi: 10.1006/jmbi.2000.3748.
Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.
大肠杆菌NADPH-亚硫酸盐还原酶（SiR）是一种780 kDa的多聚体血黄素蛋白，由八个α亚基（SiR-FP）和四个β亚基（SiR-HP）组成，催化亚硫酸盐六电子还原为硫化物。每个β亚基包含一个Fe4S4簇和一个丝氨酸亚铁血红素，每个α亚基结合一个FAD和一个FMN作为辅基。FAD从NADPH获取电子，FMN将电子转移到β亚基的金属中心以进行亚硫酸盐还原。我们在此报告SiR-FP60的1.94 Å X射线结构，SiR-FP60是SiR-FP的重组单体片段，它结合FAD和FMN并保留天然蛋白质的催化特性。该结构可分为三个结构域。酶的羧基末端部分由结合FAD的反平行β桶和结合NADPH的经典吡啶二核苷酸结合折叠变体组成。这两个结构域形成了典型的铁氧化还原蛋白NADP +还原酶家族的经典FNR样模块。与细胞色素P450还原酶的结构类似，由七个α螺旋组成的第三个结构域应该将FNR样模块连接到N末端黄素氧还蛋白样模块。在四种不同的晶体形式中，电子密度图中没有FMN结合模块，尽管对溶解晶体进行的质谱、氨基酸测序和活性实验表明蛋白质中存在一个功能模块。我们的结果清楚地表明，FNR样和FMN样模块之间的相互作用显示出比细胞色素P450还原酶更低的亲和力。FMN结合结构域的灵活性可能与细胞色素bc1的情况一样，与电子转移过程中的结构域重组有关。因此，FMN结合结构域相对于酶的其余部分的移动可能是其相对于FNR样模块和β亚基最佳定位的必要条件。
