Solution NMR Study of the Electronic Structure and Magnetic Properties of Cluster Ligation Mutants of the Four-Iron Ferredoxin from the Hyperthermophilic Archaeon Pyrococcus furiosus

详细信息    查看全文

• 作者：Luigi Calzolai ; Carol M. Gorst ; Kara L. Bren ; Zhi-Hao Zhou ; Michael W. W. Adams ; and Gerd N. La Mar
• 刊名：Journal of the American Chemical Society
• 出版年：1997
• 出版时间：October 8, 1997
• 年：1997
• 卷：119
• 期：40
• 页码：9341 - 9350
• 全文大小：243K
• 年卷期：v.119,no.40(October 8, 1997)
• ISSN：1520-5126

文摘

¹H NMR is used to characterize thesolution electronic structure and magnetic properties of thecubaneiron-sulfur clusters for wild-type (WT) and the D14C and D14S clusterligand mutants of the ferredoxin (Fd) fromthe hyperthermophilic archaeon Pyrococcus furiosus with thegoal of identifying the oxidation states of the individualiron ligated by a particular protein ligand. Sequence-specificassignments of the contact-shifted and paramagneticallyrelaxed protons for all cluster ligands are obtained in the alternatecluster oxidation states, [4Fe:4S]⁺,[4Fe:4S]²⁺,each with Cys21 and Cys48 either as free sulfhydryls or in a disulfidebond. Detailed analysis of the oxidizedcluster Fds shows that the ground state is S = 0 withsimilar population of excited S = 1 and 2 states for eachofAsp14, Ser14, or Cys14 ligated to the cluster. The reduced clusterD14C Fd exhibits pairs of Cys (Cys11,Cys17)with strong Curie, and pairs of Cys (Cys14, Cys56) with stronganti-Curie temperature dependence diagnostic ofligands to the S = ⁹/₂,2Fe^2.5+, and S = 4,2Fe^2.0+ iron pairs, respectively.Moreover, it is observed that the ligandprotons of Cys ligated to the mixed-valence pair are much moreeffectively relaxed than the Cys ligated to thediferrous pair, which is consistent with expectations for the relativespin magnetizations of the iron pair. Substitutionof Cys14 by Ser14 leads to changes in contact shift magnitudes andslopes in Curie plots that demonstrate thepresence of two electronic states that interchange the sequencepositions of the 2Fe^2.5+ and2Fe^2.0+. Similarly,replacement of Cys14 by Asp14 leads to changes in shift magnitude andCurie slope that indicate the population oftwo states for which the Asp14 is ligated to aFe^2.5+ and Cys11 is ligated to aFe^2.0+ in each state, while theothertwo ligands showed effective oxidation states midway betweenFe^2.5+ and Fe^2.0+.Thus the relative stabilization ofthe mixed-valence pair for the iron ligated by residue 14 is Asp > Ser> Cys. The localization of the mixed-valencepair to the irons ligated to Cys11 and 17 in both D14C- and D14S-Fd issignificantly stabilized for the disulfiderelative to the free sulfhydryl form of Cys21 and Cys48, and likelyreflects on a hydrogen bond between the backbonesof Cys17 and Cys21. Ligation by cyanide in reduced WT Fd yields acontact shift pattern indicative of the sameelectronic state as for the Cys14 mutant, with cyanide ligated to oneof the 2Fe^2.0+. The conversion of aFe^2.5+ligated by Asp in WT Fd to a Fe^2.0+ uponcyanide displacement of the Asp is consistent with EPR andENDORstudies (Telser, et al. J. Am. Chem. Soc.1995, 117, 5133-5140; Telser et al., submittedfor publication).