Fisher Karl, Lowe David J, Tavares Pedro, Pereira Alice S, Huynh Boi Hanh, Edmondson Dale, Newton William E
Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
J Inorg Biochem. 2007 Nov;101(11-12):1649-56. doi: 10.1016/j.jinorgbio.2007.07.037. Epub 2007 Aug 9.
Various S=3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alphaH195Q, alphaH195N, and alphaQ191K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N(2) with the alphaH195Q and alphaH195N variants, but not with the alphaQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alphaH195Q or alphaH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alphaQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze (57)Fe Mössbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alphaH195Q and alphaH195N MoFe proteins can bind N(2), but alphaQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N(2) reduction.
从野生型和变异型棕色固氮菌固氮酶钼铁蛋白中引出的各种S = 3/2电子顺磁共振（EPR）信号，似乎反映了不同质子化状态下铁钼辅因子呈现的不同构象。为了确定这些推测的质子化和构象变化是否反映催化能力，研究了具有显著不同底物还原特性的αH195Q、αH195N和αQ191K变异型钼铁蛋白（其中α亚基中第195位的组氨酸被谷氨酰胺/天冬酰胺取代，或者α-191位的谷氨酰胺被赖氨酸取代）对（特别是电子通量变化的）响应，采用停流或快速冷冻技术进行研究。在低电子通量下（野生型铁蛋白的摩尔过量3倍）进行快速冷冻EPR，在以N₂为底物开始周转的1秒内，αH195Q和αH195N变异型产生了两个基于铁钼辅因子的瞬态EPR信号，但αQ191K变异型没有。在这些条件下，未观察到任何变异型有可归因于P簇氧化的EPR信号。此外，在野生型、αH195Q或αH195N钼铁蛋白以低电子通量周转期间，也未观察到可能反映P簇氧化的较长时间的430纳米吸光度增加（通过停流分光光度法）；然而，在较高电子通量下，所有三种钼铁蛋白都出现了这种情况。αQ191K变异型即使在较高电子通量下也未出现430纳米吸光度增加。快速冷冻（⁵⁷）Fe穆斯堡尔光谱证实了在低电子通量下P簇可能不参与电子转移，该光谱清楚地显示了铁钼辅因子的还原而没有P簇氧化。由于野生型、αH195Q和αH195N钼铁蛋白可以结合N₂，但αQ195K不能，这些结果表明P簇氧化仅在N₂还原所需的高电子通量下发生。
