Conradson S D, Burgess B K, Newton W E, Di Cicco A, Filipponi A, Wu Z Y, Natoli C R, Hedman B, Hodgson K O
Department of Chemistry, Stanford University, CA 94305.
Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1290-3. doi: 10.1073/pnas.91.4.1290.
The biological N2-fixation reaction is catalyzed by the enzyme nitrogenase. The metal cluster active site of this enzyme, the iron-molybdenum cofactor (FeMoco), can be studied either while bound within the MoFe protein component of nitrogenase or after it has been extracted into N-methylformamide. The two species are similar but not identical. For example, the addition of thiophenol or selenophenol to isolated FeMoco causes its rather broad S = 3/2 electron paramagnetic resonance signal to sharpen and more closely approach the signal exhibited by protein-bound FeMoco. The nature of this thiol/selenol binding site has been investigated by using Se-K edge extended x-ray absorption fine structure (EXAFS) to study selenophenol ligated to FeMoco, and the results are reported here. EXAFS data analysis at the ligand Se-K edge was performed with a set of software, GNXAS, that provides for direct calculation of the theoretical EXAFS signals and least-squares fits to the experimental data. Data analysis results show definitively that the selenol (and by inference thiol) binds to Fe at a distance of 2.4 A. In contrast, unacceptable fits are obtained with either Mo or S as the liganded atom (instead of Fe). These results provide quantitative details about an exchangeable thiol/selenol binding site on FeMoco in its isolated, solution state and establish an Fe atom as the site of this reaction. Furthermore, the utility of ligand-based EXAFS as a probe of coordination in polynuclear metal clusters is demonstrated.
生物固氮反应由固氮酶催化。该酶的金属簇活性位点，即铁钼辅因子（FeMoco），可以在其结合于固氮酶的钼铁蛋白组分内时进行研究，也可以在将其提取到N - 甲基甲酰胺后进行研究。这两种物质相似但不完全相同。例如，向分离出的FeMoco中添加苯硫酚或苯硒酚会使其相当宽的S = 3/2电子顺磁共振信号变尖锐，并更接近蛋白质结合的FeMoco所呈现的信号。通过使用硒K边扩展X射线吸收精细结构（EXAFS）来研究与FeMoco连接的苯硒酚，对该硫醇/硒醇结合位点的性质进行了研究，结果在此报告。在配体硒K边的EXAFS数据分析是使用一套名为GNXAS的软件进行的，该软件可直接计算理论EXAFS信号并对实验数据进行最小二乘法拟合。数据分析结果明确表明，硒醇（由此推断硫醇）以2.4埃的距离与铁结合。相比之下，以钼或硫作为配位原子（而非铁）进行拟合时得到的结果不可接受。这些结果提供了关于处于分离溶液状态的FeMoco上可交换硫醇/硒醇结合位点的定量细节，并确定铁原子为该反应的位点。此外，还证明了基于配体的EXAFS作为多核金属簇中配位探针的实用性。
