Crystal Structure and Stability Studies of C77S HiPIP: A Serine Ligated [4Fe-4S] Cluster ² 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 43210 ReceiVed September 18, 2001; ReVised Manuscript ReceiVed NoVember 7, 2001 ABSTRACT: The crystal structure of Chromatium Vinosum C77S HiPIP has been determined and is compared with that of wild type. This is the first reported crystal structure of a Ser ligated [4Fe-4S] cluster and reveals a 0.11 Å shortening of the Fe-O bond (relative to Fe-S), but only minor structural alterations of the 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 of solvent access to the Fe-S cluster; however, there is no significant change in cluster solvation of the C77S mutant relative to WT HiPIP. Ser ligation ultimately results in decreased cluster stability due to increased sensitivity to proton mediated degradation. HiPIPs 1 are small [4Fe-4S] 3+/2+ cluster-containing proteins that are thought to be involved in electron-transfer reactions in photosynthetic bacteria. In some species, HiPIPs can act as an electron donor to the photosynthetic reaction center, interacting via surface hydrophobic patches (1). HiPIPs display a unique range of positive reduction potential of +50 to +450 mV (2), whereas those from low potential [4Fe- 4S] proteins are in the range of -100 to -650 mV (3). Much effort has been expended on efforts to alter the reduction potential of HiPIPs by introduction of point mutations at residue sites defining the hydrophobic core surrounding the [4Fe-4S] cluster. However, rather than perturbing the reduc- tion potential significantly, these mutations had the effect of decreasing cluster stability, and so the aromatic side-chains surrounding the cluster appear to protect the [4Fe-4S] cluster from hydrolytic attack rather than to modulate reduction potential (3-5). Such considerations have also led to investigations of cluster assembly and disassembly pathways (6-8) that are relevant in the context of iron sensing Fe-S proteins and mechanisms of cellular iron homeostasis (9). The majority of known [4Fe-4S] proteins contain clusters coordinated by four cysteines. Aconitase provided the first example of noncysteinyl coordination, in which one of the ligands is a solvent oxygen (10). Other examples include Ni-Fe hydrogenase from DesulfoVibrio gigas with a [4Fe- 4S] cluster ligated by histidine (11), and Pyrococcus furiosus ferredoxin with a [4Fe-4S] cluster ligated by an aspartate (12). As in the case of aconitase, the oxygen ligated iron in P. furiosus ferredoxin can be lost thereby generating a [3Fe-4S] cluster (13, 14). Site-directed mutagenesis is now commonly applied to mutations of cluster-bound cysteines in an attempt to identify or alter the ligands to an Fe-S cluster. In some instances, it has been found that the isosteric serine can substitute for cysteine as an unnatural ligand to the cluster. However, due to the low success rate, decreased stability of the cluster, and lack of natural examples, it appears that cysteine is significantly favored over serine. Indeed, for Azotobacter Vinelandii ferredoxin I, protein rearrangement resulting in remote cysteinyl ligation is preferred over coordination to a serine substituted in place of a ligating cysteine (15). Although there are no examples of natural serine coordination to canonical Fe-S clusters, serine has been identified as a ligand to the molybdenum center of dimethyl sulfoxide reductase (16), and a serine of the oxidized nitrogenase molybdenum-iron protein P-cluster may serve an auxiliary role by providing additional coordination to one of the iron atoms (17). The X-ray crystal structures of two classes of Fe-S protein have previously been reported following the introduction of a Cys to Ser ligand change, including the Fe(Cys) 4 rubre- doxin center and a [2Fe-2S] cluster (18, 19). However, no such examples exist for a [4Fe-4S] cluster. Relative to wild type (WT) protein, it has been previously shown that C77S HiPIP coordinates a less stable [4Fe-4S] cluster that is ligated by the O γ of Ser77 and is accompanied by no gross structural perturbations (20, 21). Although the NMR solution structure of C77S HiPIP has been solved (21), a detailed comparison between WT and C77S HiPIP clusters and the details of ² This work was supported by a grant from the Petroleum Research Fund, administered by the American Chemical Society (J.A.C.), the National Science Foundation, CHE-0111161 (J.A.C.), the NIH grant GM-59953 (R.H.), and the NIH Grant GM-17378 (M.S.). S.S.M. was supported by the NIH Chemistry and Biology Interface Training Program 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: cowan@chemistry.ohio-state.edu; tel: 614 292 2703, fax: 614 292 2703. # Evans Laboratory of Chemistry. ‡ Departments of Chemistry and Biochemistry. § Department of Molecular and Cellular Biochemistry. 1 Abbreviations: CCD, charge-coupled device; HiPIP, high potential iron protein; NMR, nuclear magnetic resonance; Tris, tris(hydroxy- methyl)-aminomethane; WT, wild type. 1195 Biochemistry 2002, 41, 1195-1201 10.1021/bi011811y CCC: $22.00 © 2002 American Chemical Society Published on Web 12/29/2001