Randall C R, Zang Y, True A E, Que L, Charnock J M, Garner C D, Fujishima Y, Schofield C J, Baldwin J E
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Biochemistry. 1993 Jul 6;32(26):6664-73. doi: 10.1021/bi00077a020.
Isopenicillin N synthase (IPNS) from Cephalosporium acremonium (M(r) 38,400) is an iron-containing enzyme that aerobically catalyzes the four-electron oxidative ring closure reactions of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), forming the beta-lactam and thiazolidine rings of isopenicillin N. Here, we report Fe K-edge X-ray absorption studies that provide insight into the iron coordination environment and the effect of substrate and nitric oxide binding. Our analysis reveals an iron(II) coordination environment consisting of two N/O-containing ligands at 2.01 +/- 0.02 A, three N/O ligands at 2.15 +/- 0.02 A, and one C/O scatterer at approximately 2.6-2.7 A. Three His ligands are associated with the 2.15-A shell, while an unsymmetrically chelated carboxylate is associated with a scatterer at 2.01 and at 2.6-2.7 A, a combination which is consistent with the ligand environment deduced from 1H NMR studies [Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. The remaining scatterer at 2.01 A is assigned to a coordinated solvent molecule, most likely hydroxide, which can act as the proton acceptor for the incoming substrate. ACV binding to Fe(II)IPNS evinces an Fe-S interaction at 2.35 +/- 0.02 A, indicative of the coordination of substrate cysteine thiolate to the metal center. Analysis of the Fe(II)IPNS-ACV-NO data reveals one Fe-N at 1.71 +/- 0.02 A, three Fe-(N,O) at 2.04 +/- 0.02 A, one Fe-S at 2.32 +/- 0.02 A, and one Fe-(C,O) at 2.61 +/- 0.02 A, the short Fe-N bond being derived from the binding of NO. Our EXAFS conclusions, supported by corresponding analysis of relevant model complexes, corroborate and refine the working model for the Fe(II) coordination environment developed from previous spectroscopic studies.
来自顶头孢霉（分子量38,400）的异青霉素N合酶（IPNS）是一种含铁酶，它在有氧条件下催化δ-（L-α-氨基己二酰基）-L-半胱氨酰-D-缬氨酸（ACV）的四电子氧化闭环反应，形成异青霉素N的β-内酰胺环和噻唑烷环。在此，我们报告了Fe K边X射线吸收研究，这些研究有助于深入了解铁的配位环境以及底物和一氧化氮结合的影响。我们的分析揭示了一种铁（II）配位环境，由两个距离为2.01±0.02 Å的含N/O配体、三个距离为2.15±0.02 Å的N/O配体以及一个距离约为2.6 - 2.7 Å的C/O散射体组成。三个组氨酸配体与2.15 Å的壳层相关，而一个不对称螯合的羧酸盐与2.01 Å和2.6 - 2.7 Å处的一个散射体相关，这种组合与从1H NMR研究推断出的配体环境一致[Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653 - 11659]。2.01 Å处的其余散射体被指定为一个配位溶剂分子，最有可能是氢氧根，它可以作为进入底物的质子受体。ACV与Fe(II)IPNS的结合在2.35±0.02 Å处显示出Fe - S相互作用，表明底物半胱氨酸硫醇盐与金属中心配位。对Fe(II)IPNS - ACV - NO数据的分析揭示了一个距离为1.71±0.02 Å的Fe - N、三个距离为2.04±0.02 Å的Fe - (N,O)、一个距离为2.32±0.02 Å的Fe - S以及一个距离为2.61±0.02 Å的Fe - (C,O)，短的Fe - N键源自NO的结合。我们的扩展X射线吸收精细结构（EXAFS）结论，在相关模型配合物的相应分析支持下，证实并完善了从先前光谱研究发展而来的Fe(II)配位环境的工作模型。
