Lu G, Lindqvist Y, Schneider G, Dwivedi U, Campbell W
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
J Mol Biol. 1995 May 19;248(5):931-48. doi: 10.1006/jmbi.1995.0273.
The refined crystal structures of the recombinant cytochrome b reductase fragment of corn (Zea mays) nitrate reductase, its ADP complex and the active-site mutant Cys242Ser are reported here. The native structure has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7% with root-mean-square (r.m.s) deviations from standard bond lengths and angles of 0.013 A and 2.0 degrees. The diffraction pattern of the crystals is highly anisotropic and correction of this effect lowered the crystallographic R-factor by 5% during the refinement. The structure of the enzyme co-crystallized with ADP has been solved at 2.7 A resolution and refined to an R-factor of 18.6% with r.m.s. deviations from standard bond lengths and angles of 0.014 A and 2.1 degrees. It revealed the binding site of the ADP moiety of the NADH cofactor, which is the electron donor for nitrate reduction. Based on this structure, a model of NADH at the active site of the enzyme was built and the implications for electron transfer from NADH to the flavin cofactor are discussed. The crystal structure of an active-site mutant enzyme, Cys242Ser, has been solved by difference Fourier synthesis and refined to an R-factor of 19.0% to 3.0 A resolution with standard deviations of bond lengths and angles of 0.017 A and 2.5 degrees. This structure analysis suggests that the observed decrease in catalytic activity of this mutant might be due to misalignment of the nicotinamide ring in its binding site. A model of the heme-containing domain of nitrate reductase has been built based on the X-ray structure of bovine cytochrome b5 and has been docked with the cytochrome b reductase fragment of nitrate reductase. The model of the complex contains six salt-bridges at the domain-domain interface and a hydrophobic core. In this model, His48, an invariant residue in the cytochrome b reductase family, forms an interaction with the propionic acid group of the D-ring of the heme cofactor. This group is in contact with the C-8 methyl group of the flavin ring. Residues that might influence the redox potential of the flavin cofactor are proposed and their possible role in electron transfer is discussed.
本文报道了玉米（Zea mays）硝酸还原酶重组细胞色素b还原酶片段、其ADP复合物以及活性位点突变体Cys242Ser的精细晶体结构。天然结构已在2.5 Å分辨率下精修至晶体学R因子为18.7%，与标准键长和键角的均方根（r.m.s）偏差分别为0.013 Å和2.0°。晶体的衍射图案具有高度各向异性，在精修过程中对这种效应进行校正后，晶体学R因子降低了5%。与ADP共结晶的酶的结构已在2.7 Å分辨率下解析，并精修至R因子为18.6%，与标准键长和键角的r.m.s偏差分别为0.014 Å和2.1°。它揭示了NADH辅因子的ADP部分的结合位点，NADH是硝酸盐还原的电子供体。基于该结构，构建了酶活性位点处NADH的模型，并讨论了从NADH到黄素辅因子的电子转移的影响。活性位点突变酶Cys242Ser的晶体结构已通过差值傅里叶合成解析，并在3.0 Å分辨率下精修至R因子为19.0%，键长和键角的标准偏差分别为0.017 Å和2.5°。该结构分析表明，观察到的该突变体催化活性降低可能是由于其结合位点中烟酰胺环的错位。基于牛细胞色素b5的X射线结构构建了硝酸还原酶含血红素结构域的模型，并将其与硝酸还原酶的细胞色素b还原酶片段对接。该复合物模型在结构域 - 结构域界面包含六个盐桥和一个疏水核心。在该模型中，细胞色素b还原酶家族中的不变残基His48与血红素辅因子D环的丙酸基团形成相互作用。该基团与黄素环的C - 8甲基接触。提出了可能影响黄素辅因子氧化还原电位 的残基，并讨论了它们在电子转移中的可能作用。
