Lee H I, Thrasher K S, Dean D R, Newton W E, Hoffman B M
Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
Biochemistry. 1998 Sep 22;37(38):13370-8. doi: 10.1021/bi980956a.
Wild-type nitrogenase MoFe protein shows a deep 14N electron spin-echo envelope modulation (ESEEM) arising from a nitrogen nucleus (N1) coupled to the S = 3/2 spin system of the FeMo-cofactor of the MoFe protein. A previous ESEEM study on altered MoFe proteins generated by substitutions at the alpha-195-histidine position suggested that alpha-195-histidine provides a hydrogen bond to the FeMo-cofactor but is not the source of the 14N1 modulation [DeRose et al. (1995) Biochemistry 34, 2809-2814]. This study also raised the possibility of a correlation between ESEEM spectroscopic properties and the nitrogenase phenotype. We now report ESEEM studies on altered MoFe proteins with substitutions at residues alpha-96-arginine, alpha-359-arginine, and alpha-381-phenylalanine to (i) assign the first-shell hydrogen bonding as revealed by the 14N modulation; (ii) explore the mechanistic relevance of the ESEEM signatures to nitrogenase activity; and (iii) study microscopic changes within the polypeptide environment of the FeMo-cofactor. Present ESEEM data reveals that two kinds of 14N modulations are present in wild-type MoFe protein. A new 2-dimensional procedure for high-precision analysis of the ESEEM data of the MoFe proteins shows that the deep wild-type ESEEM modulation (denoted N1) has a hyperfine-coupling constant of Aiso = 1.05 MHz and nuclear quadrupole coupling parameters of e2qQ = 2.17 MHz, eta = 0.59; the other (denoted N2) has a smaller hyperfine coupling of Aiso = approximately 0.5 MHz and e2qQ = approximately 3.5 MHz, eta = approximately 0.4. The N2 ESEEM pattern is more obvious when unmasked by substitutions that result in the loss of the deep N1 modulation. Correlations of the ESEEM properties and catalytic activities of the altered MoFe proteins suggest that (i) the side chain of the alpha-359-arginine is the source of the deep ESEEM N1 modulation; (ii) one or both of the amide nitrogens of alpha-356-glycine/alpha-357-glycine are responsible for the weak N2 modulation; (iii) substitution of the nonpolar alpha-381-phenylalanine residue, as well as substitution of either the alpha-195-histidine or alpha-359-arginine residues, can eliminate the N1 interaction with FeMo-cofactor; and (iv) ESEEM can be used to detect slight reorientations of FeMo-cofactor within its polypeptide pocket, although the mechanistic relevance of the loss or perturbation of the hydrogen-bonding interactions between FeMo-cofactor and polypeptide environment has not yet been established.
野生型固氮酶钼铁蛋白显示出由与钼铁蛋白的铁钼辅因子的S = 3/2自旋系统耦合的氮核（N1）产生的深度14N电子自旋回波包络调制（ESEEM）。先前对在α-195-组氨酸位置进行取代产生的改变的钼铁蛋白的ESEEM研究表明，α-195-组氨酸为铁钼辅因子提供氢键，但不是14N1调制的来源[德罗塞等人（1995年）《生物化学》34卷，2809 - 2814页]。这项研究还提出了ESEEM光谱性质与固氮酶表型之间存在相关性的可能性。我们现在报告对在α-96-精氨酸、α-359-精氨酸和α-381-苯丙氨酸残基处进行取代的改变的钼铁蛋白的ESEEM研究，以（i）确定由14N调制揭示的第一壳层氢键；（ii）探索ESEEM特征与固氮酶活性的机制相关性；以及（iii）研究铁钼辅因子多肽环境内的微观变化。目前的ESEEM数据表明，野生型钼铁蛋白中存在两种14N调制。一种用于高精度分析钼铁蛋白ESEEM数据的新二维程序表明，深度野生型ESEEM调制（表示为N1）具有超精细耦合常数Aiso = 1.05 MHz和核四极耦合参数e2qQ = 2.17 MHz，η = 0.59；另一种（表示为N2）具有较小的超精细耦合Aiso = 约0.5 MHz和e2qQ = 约3.5 MHz，η = 约0.4。当被导致深度N1调制丧失的取代所掩盖时，N2 ESEEM模式更明显。改变的钼铁蛋白的ESEEM性质与催化活性的相关性表明，（i）α-359-精氨酸的侧链是深度ESEEM N1调制的来源；（ii）α-356-甘氨酸/α-357-甘氨酸的一个或两个酰胺氮负责弱N2调制；（iii）非极性α-381-苯丙氨酸残基的取代，以及α-195-组氨酸或α-359-精氨酸残基的取代，都可以消除与铁钼辅因子的N1相互作用；并且（iv）ESEEM可用于检测铁钼辅因子在其多肽口袋内的轻微重新定向，尽管铁钼辅因子与多肽环境之间氢键相互作用的丧失或扰动的机制相关性尚未确定。
