Ravi N, Moore V, Lloyd S G, Hales B J, Huynh B H
Department of Physics, Rollins Research Center, Emory University, Atlanta, Georgia 30322.
J Biol Chem. 1994 Aug 19;269(33):20920-4.
The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.
从维涅兰德固氮菌LS15菌株中分离出的替代固氮酶的VFe蛋白，命名为Av1'，已通过穆斯堡尔光谱进行了研究。在4.2 K、平行于γ射线施加60毫特斯拉磁场的条件下记录的连二亚硫酸盐还原的Av1'的穆斯堡尔光谱是三个光谱成分的叠加：1）一个复杂光谱（M成分），具有归因于顺磁性FeV辅因子的磁超精细结构；2）一个成分（P成分），由三个四极双峰组成，可识别为Fe2+、D和S双峰，类似于在MoFe蛋白的P簇对中观察到的双峰；3）一个次要的（占总吸收的4%）四极双峰，归因于偶然结合的铁。观察到的Fe2+（δEQ = 2.99 mm/s，δ = 0.64 mm/s）、D（δEQ = 0.75 mm/s，δ = 0.63 mm/s）和S（δEQ = 1.2 mm/s，δ = 0.65 mm/s）铁位点在4.2 K时的参数及其温度依赖性与在传统MoFe蛋白的P簇对中观察到的非常相似。与MoFe蛋白类似，强场光谱表明这些双峰与抗磁系统相关。在去除偶然结合的铁的贡献后，在4.2 K时测定的它们的吸收强度百分比（Fe(2+)/D/S = 13.0:32.2:6.8）与MoFe蛋白中P簇对的吸收强度百分比相当。这些观察结果表明，Av1'也含有在组成和数量上与MoFe蛋白相同的P簇对；即每个分子含有两个P簇对，每个对由两个Fe2+、五个D和一个S铁位点组成。考虑到P成分对应于两个8Fe簇的52%吸收，为M成分测定的其余48%吸收与Av1'的每个分子含有两个含7Fe的FeV辅因子一致。两个P簇对都处于抗磁状态这一事实意味着在早期EPR测量中检测到的S = 3/2和S = 1/2信号与FeV辅因子相关，并表明在连二亚硫酸盐还原的Av1'中FeV辅因子存在自旋混合。
