Wijma Hein J, Macpherson Iain, Alexandre Maxime, Diederix Rutger E M, Canters Gerard W, Murphy Michael E P, Verbeet Martin Ph
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
J Mol Biol. 2006 May 12;358(4):1081-93. doi: 10.1016/j.jmb.2006.02.042. Epub 2006 Mar 6.
In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.
在粪产碱菌S-6含铜亚硝酸还原酶中，1型位点的轴向甲硫氨酸配体被替换（M150G），以使铜离子能与可能影响酶催化活性的外部配体接触。M150G的1型位点光谱（A(460)/A(600)=0.71）与天然亚硝酸还原酶的光谱（A(460)/A(600)=1.3）有显著差异。亚硝酸还原酶M150G的1型位点中点电位（相对于氢，E(M)=312(±5)mV）高于天然酶（E(M)=213(±5)mV）。M150G的催化活性（k(cat)=133(±6)s⁻¹）低于野生型亚硝酸还原酶（k(cat)=416(±10)s⁻¹）。外部配体与M150G的结合恢复了光谱特性、中点电位（E(M)<225mV）和催化活性（k(cat)=374(±28)s⁻¹）。此外，还对M150H（A(460)/A(600)=7.7，E(M)=104(±5)mV，k(cat)=0.099(±0.006)s⁻¹）和M150T（A(460)/A(600)=0.085，E(M)=340(±5)mV，k(cat)=126(±2)s⁻¹）变体进行了表征。晶体结构表明，配体通过取代甲硫氨酸62发挥变构效应，甲硫氨酸62移动到与M150G突变空出位置的铜结合。重构的1型位点在其他方面具有未改变的几何结构。去除内源性配体可在氧化还原酶中引入变构控制这一观察结果表明含铜氧化还原位点具有结构和功能灵活性的潜力。
