research papers 1582 Gonza Âlez et al.  R. fermentans HiPIP Acta Cryst. (2003). D59, 1582±1588 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Structure of Rhodoferax fermentans high-potential iron±sulfur protein solved by MAD Ana Gonza Âlez, a Stefano Benini b and Stefano Ciurli c * a Stanford Synchrotron Radiation Laboratory, 2575 Sand Hill Road MS99, Menlo Park, CA 94025, USA, b York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, England, and c Department of Agro-Environmental Science and Technology, University of Bologna, Viale Fanin 40, I-40100 Bologna, Italy Correspondence e-mail: stefano.ciurli@unibo.it # 2003 International Union of Crystallography Printed in Denmark ± all rights reserved The crystal structure of Rhodoferax fermentans high-potential iron protein (HiPIP) has been solved by MAD methods using the anomalous signal from the Fe atoms in the [Fe 4 S 4 ] cluster present in the protein and re®ned to a resolution of 1.45 A Ê . The peptide chain is well de®ned except in the N- and C-terminal areas. The structure of the protein reveals the presence of three helical fragments, a small -sheet and several turns, with the [Fe 4 S 4 ] cluster being located close to a surface patch containing several well conserved aromatic residues. The protein fold is very similar to the structures of other known HiPIPs, especially in the region proximal to the [Fe 4 S 4 ] cluster, while the largest differences are observed on the opposite side of the protein, which is rich in positive charges and has no sequential homology to other HiPIP families. Received 7 May 2003 Accepted 30 June 2003 PDB Reference: R. fermen- tans HiPIP, 1hlq, r1hlqsf. 1. Introduction HiPIPs (high-potential iron±sulfur proteins) are a class of small (8±10 kDa) proteins containing a cubane [Fe 4 S 4 ] cluster bound to the protein backbone by four Fe±S Cys bonds (Carter, 2001). HiPIPs are peculiar among iron±sulfur proteins because of the high reduction potential at which the metal cluster exchanges electrons (from +50 to +450 mV; Meyer et al., 1983) using the [Fe 4 S 4 ] 3+/2+ couple (Carter et al., 1972; Middleton et al., 1980). HiPIPs have been extensively inves- tigated as electron-transfer models (Rawlings et al. , 1976; Mizrahi et al., 1976, 1980; Mizrahi & Cusanovich, 1980; Aprahamian & Feinberg, 1981; Meyer et al., 1983, 1995; Przysiecki et al., 1985; Babini et al., 2000) and their electronic, spectroscopic and redox properties are well characterized (Bertini et al., 1995; Capozzi et al., 1998). Even though the structures of several HiPIPs have been determined (Carter et al., 1974; Freer et al., 1975; Breiter et al., 1991; Rayment et al., 1992; Benning et al., 1994; Kerfeld et al., 1998; Parisini et al., 1999; Nogi et al., 2000, Liu et al., 2002), their biological func- tion is still a matter of discussion. HiPIPs were initially shown to participate in direct (Hooper & Di Spirito, 1985) and indirect (Tedro et al. , 1977; Fukumori & Yamanaka, 1979; Kusano et al. , 1992) substrate-oxidation reactions in purple sulfur bacteria. In recent years, attention has been directed towards the involvement of HiPIPs in the respiratory (Bonora et al., 1999) or photosynthetic machinery of phototrophic bacteria. This possibility is supported by the fact that HiPIPs are abundant in most species of purple phototrophic bacteria that lack cytochrome c 2 (Bartsch, 1991). The latter are well established electron carriers in the cyclic