Refined Structures of Oxidized Flavodoxin from Anacystis nidulans Catherine L. Drennan, Katherine A. Pattridge, Christian H. Weber, Anita L. Metzger, David M. Hoover and Martha L. Ludwig* Department of Biological Chemistry and Biophysics Research Division, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109, USA Flavodoxin from Anacystis nidulans (Synechococcus PCC 7942) was the ®rst member of the ¯avodoxin family to be characterized, and is the structural prototype for the ``long-chain'' ¯avodoxins that have molecular masses of 20 kDa. Crystal structure analyses and re®nements of three orthorhombic forms of oxidized A. nidulans ¯avodoxin are reported, and salient features of the fold and the FMN binding site are compared with other ¯avodoxins. The structure of form I (wild-type: P2 1 2 1 2 1 , a 57.08 A Ê , b 69.24 A Ê , c 45.55 A Ê ), determined initially by multiple isomorphous replacement, has been re®ned to R 0.183 and R free 0.211 for data from 10.0 to 1.7 A Ê resolution. Structures of form II (wild-type: P2 1 2 1 2 1 , a 60.05 A Ê , b 65.85 A Ê , c 51.36 A Ê ) and form III (Asn58Gly: P2 1 2 1 2 1 , a 51.30 A Ê , b 59.15 A Ê , c 94.44 A Ê ) have been determined by molecular replacement and re®ned versus data to 2.0 A Ê and 1.85 A Ê , respectively; the R values for forms II and III are 0.147 and 0.150. Changes in the molecular contacts that produce the alternative packings in these crystalline forms are analyzed. Deletion of the Asn side-chain in the mutant Asn58Gly removes an intermolecular stacking interaction and allows the alternative packing found in form III crystals. The functionally important 50's loop of the FMN binding site is less restrained by intermo- lecular contacts in these crystals but maintains the same conformation as in oxidized wild type protein. The structures reported here provide the starting point for structure-function studies of the reduced states and of mutants, described in the accompanying paper. # 1999 Academic Press Keywords: ¯avodoxin; FMN binding; redox potential; crystal packing *Corresponding author Introduction Flavodoxins are the smallest members of the ¯a- voprotein family, with molecular masses ranging from 14 to 23 kDa. Flavodoxin from the cyanobac- terium Anacystis nidulans (or Synechococcus PCC 7942), the subject of this study, was the ®rst of these electron transfer proteins to be characterized (Smillie, 1965). Subsequently, ¯avodoxins have been isolated from almost 20 other species of microorganisms, including two eukaroytes, Chon- drus crispus and Chlorella fusca. Bacterial ¯avodox- ins are surrogates for ferredoxins in a variety of electron transfer reactions at low potential (Knight & Hardy, 1966; Mayhew & Ludwig, 1975; Mayhew & Tollin, 1992; Smillie, 1965). A speci®c require- ment for ¯avodoxin (nifF) as the electron donor to nitrogenase has been demonstrated by analysis of mutations in the nif gene cluster of Klebsiella pneu- moniae (Deistung et al., 1985; Nieva-Gomez et al., 1980). In Escherichia coli, ¯avodoxin ®lls a special role as the physiological electron donor for the reactivation or priming of several enzymes, includ- ing cobalamin-dependent methionine synthase (Fujii et al., 1977; Hoover & Ludwig, 1997), pyru- vate formate lyase (Blaschkowski et al., 1982), and anaerobic ribonucleotide reductase (Bianchi et al., 1993). Flavodoxins are not found as independent proteins in mammalian systems, but are incorpor- ated as domains in larger redox proteins such as cytochrome P450 reductase (Wang et al., 1997). The size, stability, and ease of isolation of ¯avo- doxins have made these proteins ideal subjects for Present addresses: C. L. Drennan, Dept. Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 USA; D. M. Hoover, Protein Structure Section, National Cancer Institute, Frederick, MD 21702, USA. E-mail address of the corresponding author: ludwig@biop.umich.edu Article No. jmbi.1999.3151 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 711±724 0022-2836/99/480711±14 $30.00/0 # 1999 Academic Press