Tocheva Elitza I, Eltis Lindsay D, Murphy Michael E P
Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver BC, V6T 1Z3, Canada.
Biochemistry. 2008 Apr 15;47(15):4452-60. doi: 10.1021/bi7020537. Epub 2008 Mar 22.
The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.
利用晶体学和稳态动力学研究了粪产碱菌S-6（AfNiR）中含铜异化亚硝酸盐还原酶与五个小分子之间的相互作用。结构研究表明，每个小分子都与AfNiR氧化态的2型催化铜相互作用。三个小分子（甲酸、乙酸和硝酸盐）通过至少有两个氧原子与2型铜原子进行双齿配位来模拟底物。这三种阴离子以与亚硝酸盐相同的不对称双齿方式与铜离子结合。与它们对酶的弱抑制作用（Ki>50 mM）一致，这些AfNiR-抑制剂复合物中的Cu-O距离比AfNiR-亚硝酸盐复合物中观察到的距离长约0.15 Å。每种抑制剂的结合模式部分由与活性位点残基Ile257侧链的空间相互作用决定。此外，Asp98的侧链是一个保守残基，与2型铜结合的亚硝酸盐和一氧化氮形成氢键，它要么无序，要么远离抑制剂。乙酸和甲酸以混合方式抑制AfNiR，这与AfNiR-乙酸复合物中第二个乙酸结合位点的出现一致，该位点阻碍了对2型铜的接近。第四个小分子一氧化二氮以侧面结合的方式与氧化态金属结合，类似于一氧化氮与还原态铜的结合方式。然而，一氧化二氮与金属的结合距离更远。第五个小分子叠氮化物对AfNiR还原亚硝酸盐的抑制作用最强（Kic = 2.0 +/- 0.1 mM）。该配体以端对端的方式与2型铜中心结合，Cu-N c距离约为2 Å，并且是唯一与Asp98形成氢键的抑制剂。总体而言，这些数据证实了Asp98和Ile257在区分底物与其他小阴离子方面的作用。
