X-ray absorption studies of the ferrous active site of isopenicillin N synthase and related model complexes

Abstract

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.

Anaerobic Crystallisation of an Isopenicillin N Synthase · Fe(II) · Substrate Complex Demonstrated by X‐Ray Studies

“…The overall accuracy given for the two distances includes systematic errors such as accuracy of phase shifts and is given to be on the conservative end. These results correlate well with previous solution studies [ 5 ] on the Cephalosporium ncremonium IPNS . Fe(I1) .…”

Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans

“…It must be concluded that the single imidazole per metal atom in the HuHF ferroxidase site does not contribute significantly to the EXAFS. This is somewhat unexpected, but it must be noted that examples where even three imidazole ligands to an iron atom do not give a detectable outer-shell contribution to the EXAFS have been reported [38]. The intensity of those EXAFS features has also been found to depend on the orientation of the imidazole ligand with respect to the metal-donor atom bond [36].…”

XANES Study of the Carboxylate Binding Mode in Two Pterin Hydroxylases

“…The intensity of the pre-edge feature increases with the loss of inversion symmetry caused by a decreasing coordination number. This has been studied intensively in model systems [15] and is well-established in the analysis of iron proteins [16] [17]. Recently, the richness of the information of the pre-edge peak has been unveiled in Fe catalysts using novel high-resolution K b detected XANES [18].…”