title: Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins.
abstract: A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of MnIIand FeII. Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess MnIIpromotes heterobimetallic cofactor assembly. In the absence of FeII, R2c cooperatively binds MnIIat both metal sites, whereas R2lox does not readily bind MnIIat either site. Heterometallic cofactor assembly is favored at substoichiometric FeIIconcentrations in R2lox. FeIIand MnIIlikely bind to the protein in a stepwise fashion, with FeIIbinding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of MnIIby FeIIat site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular MnII/FeIIconcentrations in the host organisms from which they were isolated.
authors.affiliation: Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kutin
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Vivek
authors.initials: V
authors.lastname: Srinivas
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Fritz
authors.affiliation: Institut für Experimentalphysik, Freie Universität Berlin, D-14195 Berlin, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Kositzki
authors.affiliation: Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Hannah S
authors.initials: HS
authors.lastname: Shafaat
authors.affiliation: Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: James
authors.initials: J
authors.lastname: Birrell
authors.affiliation: Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Bill
authors.affiliation: Institut für Experimentalphysik, Freie Universität Berlin, D-14195 Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
authors.affiliation: Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lubitz
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; Department of Chemistry, Stanford University, Stanford, CA 94305, United States. Electronic address: hogbom@dbb.su.se.
authors.forename: Martin
authors.initials: M
authors.lastname: Högbom
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: griese@dbb.su.se.
authors.forename: Julia J
authors.initials: JJ
authors.lastname: Griese
authors.affiliation: Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, D-45470 Mülheim an der Ruhr, Germany; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia. Electronic address: nicholas.cox@cec.mpg.de.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Cox
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056506:Geobacillus / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics; D016930:Saccharopolyspora / Q000737:chemistry*; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012996:Solutions; D008345:Manganese; D007501:Iron; D010088:Oxidoreductases; D012264:Ribonucleotide Reductases
keywords: Di-metal carboxylate protein; EPR spectroscopy; Ferritin; Mössbauer spectroscopy; Ribonucleotide reductase; X-ray crystallography