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Crystal Structure and Stability Studies

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Crystal Structure and Stability Studies of C77S HiPIP: A Serine Ligated [4Fe-4S]Cluster†Sheref S. Mansy,#Yong Xiong,‡Craig Hemann,§Russ Hille,§M. Sundaralingam,*,‡and J. A. Cowan*,#EVans Laboratory of Chemistry, Ohio State UniVersity, 100 West 18th AVenue, Columbus, Ohio 43210,Department of Molecular and Cellular Biochemistry, Ohio State UniVersity, 1645 Neil AVenue, Columbus, Ohio 43210,and Departments of Chemistry and Biochemistry, Macromolecular Structure Center, Ohio State UniVersity,1060 Carmack Road, Columbus, Ohio 43210ReceiVed September 18, 2001; ReVised Manuscript ReceiVed NoVember 7, 2001ABSTRACT: The crystal structure of Chromatium Vinosum C77S HiPIP has been determined and is comparedwith that of wild type. This is the first reported crystal structure of a Ser ligated [4Fe-4S] cluster andreveals a 0.11 Å shortening of the Fe-O bond (relative to Fe-S), but only minor structural alterations ofthe overall tertiary structure. Coordination changes are corroborated by resonance Raman spectroscopy.Comparison of the crystal and solution structures for HiPIPs identifies Phe48 as the main controller ofsolvent access to the Fe-S cluster; however, there is no significant change in cluster solvation of theC77S mutant relative to WT HiPIP. Ser ligation ultimately results in decreased cluster stability due toincreased sensitivity to proton mediated degradation.HiPIPs1are small [4Fe-4S]3+/2+cluster-containing proteinsthat are thought to be involved in electron-transfer reactionsin photosynthetic bacteria. In some species, HiPIPs can actas an electron donor to the photosynthetic reaction center,interacting via surface hydrophobic patches (1). HiPIPsdisplay a unique range of positive reduction potential of +50to +450 mV (2), whereas those from low potential [4Fe-4S] proteins are in the range of -100 to -650 mV (3). Mucheffort has been expended on efforts to alter the reductionpotential of HiPIPs by introduction of point mutations atresidue sites defining the hydrophobic core surrounding the[4Fe-4S] cluster. However, rather than perturbing the reduc-tion potential significantly, these mutations had the effectof decreasing cluster stability, and so the aromatic side-chainssurrounding the cluster appear to protect the [4Fe-4S] clusterfrom hydrolytic attack rather than to modulate reductionpotential (3-5). Such considerations have also led toinvestigations of cluster assembly and disassembly pathways(6-8) that are relevant in the context of iron sensing Fe-Sproteins and mechanisms of cellular iron homeostasis (9).The majority of known [4Fe-4S] proteins contain clusterscoordinated by four cysteines. Aconitase provided the firstexample of noncysteinyl coordination, in which one of theligands is a solvent oxygen (10). Other examples includeNi-Fe hydrogenase from DesulfoVibrio gigas with a [4Fe-4S] cluster ligated by histidine (11), and Pyrococcus furiosusferredoxin with a [4Fe-4S] cluster ligated by an aspartate(12). As in the case of aconitase, the oxygen ligated ironin P. furiosus ferredoxin can be lost thereby generating a[3Fe-4S] cluster (13, 14).Site-directed mutagenesis is now commonly applied tomutations of cluster-bound cysteines in an attempt to identifyor alter the ligands to an Fe-S cluster. In some instances, ithas been found that the isosteric serine can substitute forcysteine as an unnatural ligand to the cluster. However, dueto the low success rate, decreased stability of the cluster,and lack of natural examples, it appears that cysteine issignificantly favored over serine. Indeed, for AzotobacterVinelandii ferredoxin I, protein rearrangement resulting inremote cysteinyl ligation is preferred over coordination to aserine substituted in place of a ligating cysteine (15).Although there are no examples of natural serine coordinationto canonical Fe-S clusters, serine has been identified as aligand to the molybdenum center of dimethyl sulfoxidereductase (16), and a serine of the oxidized nitrogenasemolybdenum-iron protein P-cluster may serve an auxiliaryrole by providing additional coordination to one of the ironatoms (17).The X-ray crystal structures of two classes of Fe-S proteinhave previously been reported following the introduction ofa Cys to Ser ligand change, including the Fe(Cys)4rubre-doxin center and a [2Fe-2S] cluster (18, 19). However, nosuch examples exist for a [4Fe-4S] cluster. Relative to wildtype (WT) protein, it has been previously shown that C77SHiPIP coordinates a less stable [4Fe-4S] cluster that is ligatedby the Oγof Ser77 and is accompanied by no gross structuralperturbations (20, 21). Although the NMR solution structureof C77S HiPIP has been solved (21), a detailed comparisonbetween WT and C77S HiPIP clusters and the details of†This work was supported by a grant from the Petroleum ResearchFund, administered by the American Chemical Society (J.A.C.),the National Science Foundation, CHE-0111161 (J.A.C.), the NIHgrant GM-59953 (R.H.), and the NIH Grant GM-17378 (M.S.). S.S.M.was supported by the NIH Chemistry and Biology Interface TrainingProgram at Ohio State University (GM08512-03).* Address correspondence to Professor J. A. Cowan at the Depart-ment of Chemistry, Ohio State University, 100 West 18th Ave.,Columbus, OH 43210. E-mail: [email protected]; tel:614 292 2703, fax: 614 292 2703.#Evans Laboratory of Chemistry.‡Departments of Chemistry and Biochemistry.§Department of Molecular and Cellular Biochemistry.1Abbreviations: CCD, charge-coupled device; HiPIP, high potentialiron protein; NMR, nuclear magnetic resonance; Tris, tris(hydroxy-methyl)-aminomethane; WT, wild type.1195Biochemistry 2002, 41, 1195-120110.1021/bi011811y CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 12/29/2001cluster coordination was precluded as a result of theparamagnetism of the cluster. X-ray crystallography canprovide additional and more accurate structural details ofthe cluster environment. Here we report the first X-ray crystalstructure of a serine oxygen ligated [4Fe-4S] cluster.Comparisons of the cluster and surrounding aromatic residuesof Chromatium Vinosum C77S HiPIP and WT HiPIP arediscussed.MATERIALS AND METHODSProtein Purification and Crystallization. Preparation andinitial purification procedures were as previously described(20, 22). Further purification was achieved by gel filtration(G-75, Pharmacia) with 10 mM Tris-HCl, pH 8.0, as therunning buffer. C77S HiPIP was concentrated via ultrafil-tration (Amicon) to 1.1 mM. Hollow


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