Cysteine Ligand Swapping on a Deletable Loop of the [2Fe-2S] Ferredoxin from Clostridium pasteurianum † Marie-Pierre Golinelli, ‡ Leah A. Akin, § Brian R. Crouse, § Michael K. Johnson, § and Jacques Meyer* ,‡ Me ´ talloprote ´ ines, De ´ partement de Biologie Mole ´ culaire et Structurale, CEA-Grenoble, 38054 Grenoble, France, and Department of Chemistry and Center for Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602 ReceiVed February 21, 1996; ReVised Manuscript ReceiVed May 7, 1996 X ABSTRACT: The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, and 60). In previous investigations, a combination of site-directed mutagenesis and of spectroscopic techniques showed that cysteines 11, 56, and 60 are ligands of the [2Fe-2S] cluster in the wild type protein and that cysteine 14 is not, but the status of cysteine 24 remained unclear. New mutated forms of this ferredoxin have been obtained and characterized. The data show that cysteine 24 is a ligand of the cluster in the wild type protein. When cysteine 24 is mutated into alanine, it is replaced as a cluster ligand by cysteine 14. The fourth ligand of the cluster can also be a cysteine residue newly introduced in position 16 when both cysteines 14 and 24 are replaced by alanine. These results suggest that the region encompassing cysteines 14 and 24 is a solvent-exposed flexible loop, in agreement with structure predictions. A number of nondeleterious deletions of variable length (3-14 residues) have been performed in the region of residues 17-32. The deletions were found to modify only marginally the spectroscopic properties of the [2Fe- 2S] cluster but resulted in variations of its redox potential over a range of nearly 100 mV. This is the first instance of ligand swapping in a [2Fe-2S] protein, and the first time in any ferredoxin that a large loop has been excised from the structure without preventing the assembly of the iron-sulfur chromophore. Some of the molecular variants described here also highlight the similarities between the C. pasteurianum [2Fe-2S] ferredoxin and the 25 kDa subunit of the proton-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans. Iron-sulfur clusters of the [2Fe-2S] type occur in a wide range of proteins involved in electron transfer, catalysis of redox reactions (Cammack, 1992; Matsubara & Saeki, 1992), assembly of metallic active sites (Fu et al., 1994), or regulation of gene expression (Hidalgo & Demple, 1994). They are present either as sole prosthetic groups in small redox proteins (Matsubara & Saeki, 1992) or as components of proteins containing multiple redox sites (Cammack, 1992; Johnson, 1994). [2Fe-2S] proteins are remarkable because of the diverse distributions of the cluster ligands in their sequences. From this viewpoint, the [2Fe-2S] ferredoxin from the nitrogen-fixing saccharolytic anaerobe Clostridium pasteurianum (Cp 1 2Fe Fd) has long been unique and has therefore attracted much interest (Meyer et al., 1986a,b; Meyer, 1988, 1993; Fu et al., 1992). The gene encoding this protein has been expressed in Escherichia coli (Fujinaga & Meyer, 1993), and all of its five cysteine residues in positions 11, 14, 24, 56, and 60 of the 102-amino acid sequence have been mutated into serine or alanine (Fujinaga et al., 1993; Meyer et al., 1994). These data have provided the unequivocal assignment of cysteines 11, 56, and 60 as ligands of the cluster, the characterization of serine-ligated [2Fe-2S] clusters (Fujinaga et al., 1993; Meyer et al., 1994), and the uncovering in the latter of unprecedented magnetic properties (Crouse et al., 1995). The preparation and characterization of a number of new molecular variants of the Cp 2Fe Fd are reported here. For each mutant, the excited state electronic structure and the ground state vibrational properties of the S ) 0 [2Fe-2S] 2+ clusters in the oxidized proteins have been investigated by UV-visible absorption and resonance Raman (RR) spec- troscopies, respectively. The excited and ground state electronic properties of the S ) 1 / 2 [2Fe-2S] + cluster in the reduced proteins have been investigated by variable-tem- perature magnetic circular dichroism (VTMCD) and EPR spectroscopies, respectively. These data have allowed the identification of all ligands of the cluster in the WT protein and in various mutants thereof. A new case of ligand swapping has been disclosed, an occurrence that was unprecedented in [2Fe-2S] ferredoxins. Furthermore, in the region of the two exchangeable ligands, cysteines 14 and 24, deletions of up to 14 amino acids can be performed without considerably destabilizing the protein. The effects of these mutations on the structural, electronic, and redox properties of Cp 2Fe Fd are discussed. The numerous molecular variants described here also enlighten relationships in sequence and active site structure between Cp 2Fe Fd and other [2Fe-2S] proteins. † This work was supported by a grant from the National Institutes of Health (GM51962 to M.K.J.). * Address for correspondence: Jacques Meyer, DBMS-Me ´tallopro- te ´ines, CEA-Grenoble, 38054 Grenoble, France. Fax: 76 88 58 72. E-mail: jac@ebron.ceng.cea.fr. ‡ DBMS-Me ´talloprote ´ines. § University of Georgia. X Abstract published in AdVance ACS Abstracts, June 15, 1996. 1 Abbreviations: Fd, ferredoxin; Cp, Clostridium pasteurianum; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; Pd, Paracoccus denitrificans; PCR, polymerase chain reaction; RR, resonance Raman; VTMCD, variable-temperature magnetic circular dichroism; WT, wild type. 8995 Biochemistry 1996, 35, 8995-9002 S0006-2960(96)00428-X CCC: $12.00 © 1996 American Chemical Society