Dilworth M J, Fisher K, Kim C H, Newton W E
Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg 24061, USA.
Biochemistry. 1998 Dec 15;37(50):17495-505. doi: 10.1021/bi9812017.
Studies of the substrate-reducing capabilities of an altered nitrogenase MoFe protein (alpha-195(Gln) instead of alpha-195(His)) from a mutant of Azotobacter vinelandii show, contrary to an earlier report [Kim, C.-H., Newton, W. E., and Dean, D. R. (1995) Biochemistry 34, 2798-2808], that the alpha-195(Gln) MoFe protein can reduce N2 to NH3 but at a rate that is <2% of that of the wild type. The extent of effective binding of N2 by this altered MoFe protein, as monitored by the inhibition of H2 evolution, is markedly increased as temperature is lowered but virtually eliminated at 45 degreesC. This inhibition of H2 evolution results in an increase in the ATP:2e- ratio, i.e., the number of molecules of MgATP hydrolyzed for each electron pair transferred to substrate, from ca. 5 (the wild-type level) at 45 degreesC to nearly 25 at 13 degreesC. Like wild-type nitrogenase, the N2 inhibition of H2 evolution reaches a maximum at an Fe protein:MoFe protein molar ratio of ca. 2.5, suggesting that a highly reduced enzyme may not be necessary for N2 binding. N2 binding to the alpha-195(Gln) MoFe protein retains a hallmark of the wild type by producing HD under a mixed N2/D2 atmosphere. The rate of HD production and the fraction of total electron flow allocated to HD are similar to those for wild-type nitrogenase under the same conditions. However, the electrons forming HD do not come from those normally producing NH3 (as occurs in the wild type) but are equivalent to those whose evolution as H2 had been inhibited by N2. N2 also inhibits C2H2 reduction catalyzed by the alpha-195(Gln) nitrogenase. This inhibition is relieved by added H2, resulting in a lowering of the elevated ATP:2e- ratio to that found under Ar. With solutions of NaCN, which contain both the substrate, HCN, and the inhibitor, CN-, reduction of HCN is not impaired with the alpha-195(Gln) nitrogenase, but the inhibition by CN- of total electron flow to substrate, which is observed with the wild-type MoFe protein, is completely absent. Unlike that of the catalyzed reduction of H+, HCN, or C2H2, the extent of azide reduction to either N2 or N2H4 is markedly decreased (to 5-7% of that of the wild type) with the alpha-195(Gln) nitrogenase. Azide, like N2, inhibits H2 evolution and increases the ATP:2e- ratio. Both effects are freely reversible and abolished by CO. Added D2 does not relieve either effect, implying that N2 produced from N3- is not the inhibitory species. The correlation between the extremely low rates of reduction for both N2 and azide by the alpha-195(Gln) nitrogenase and their common ability to inhibit H2 evolution suggests that alpha-histidine-195 may be an important proton conductor to the FeMo cofactor center and specifically required for reduction of these two substrates.
对来自维涅兰德固氮菌突变体的一种改变的固氮酶钼铁蛋白（α-195（谷氨酰胺）而非α-195（组氨酸））的底物还原能力的研究表明，与早期报告[Kim, C.-H., Newton, W. E., and Dean, D. R. (1995) Biochemistry 34, 2798 - 2808]相反，α-195（谷氨酰胺）钼铁蛋白能够将N₂还原为NH₃，但速率不到野生型的2%。通过H₂释放的抑制来监测，这种改变的钼铁蛋白对N₂的有效结合程度随着温度降低而显著增加，但在45℃时几乎消失。这种对H₂释放的抑制导致ATP:2e⁻比值增加，即每转移到底物的一对电子水解的MgATP分子数，从45℃时约5（野生型水平）增加到13℃时接近25。与野生型固氮酶一样，N₂对H₂释放的抑制在铁蛋白:钼铁蛋白摩尔比约为2.5时达到最大值，这表明高度还原的酶对于N₂结合可能不是必需的。在混合N₂/D₂气氛下，N₂与α-195（谷氨酰胺）钼铁蛋白的结合通过产生HD保留了野生型的一个特征。在相同条件下，HD产生的速率和分配到HD的总电子流分数与野生型固氮酶相似。然而，形成HD的电子并非来自通常产生NH₃的那些电子（如野生型那样），而是等同于其作为H₂的释放被N₂抑制的那些电子。N₂也抑制α-195（谷氨酰胺）固氮酶催化的C₂H₂还原。添加H₂可缓解这种抑制，导致升高的ATP:2e⁻比值降低至在氩气下发现的值。对于含有底物HCN和抑制剂CN⁻的NaCN溶液，α-195（谷氨酰胺）固氮酶对HCN的还原不受影响，但野生型钼铁蛋白中观察到的CN⁻对流向底物的总电子流的抑制完全不存在。与催化还原H⁺、HCN或C₂H₂不同，α-195（谷氨酰胺）固氮酶将叠氮化物还原为N₂或N₂H₄的程度显著降低（至野生型水平的5 - 7%）。叠氮化物与N₂一样，抑制H₂释放并增加ATP:2e⁻比值。这两种效应都是可逆的，且被CO消除。添加D₂不能缓解任何一种效应，这意味着由N₃⁻产生的N₂不是抑制性物质。α-195（谷氨酰胺）固氮酶对N₂和叠氮化物的极低还原速率与其共同的抑制H₂释放能力之间的相关性表明，α-组氨酸-195可能是铁钼辅因子中心的重要质子导体，并且是还原这两种底物所特别需要的。
