title: Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.
abstract: Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.
authors: Free University Berlin, Institute of Experimental Physics, 14195 Berlin, Germany.
authors: Kajsa G V
authors: KGV
authors: Sigfridsson
authors: Free University Berlin, Institute of Experimental Physics, 14195 Berlin, Germany.
authors: Petko
authors: P
authors: Chernev
authors: Free University Berlin, Institute of Experimental Physics, 14195 Berlin, Germany.
authors: Nils
authors: N
authors: Leidel
authors: Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden.
authors: Ana
authors: A
authors: Popović-Bijelić
authors: Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden.
authors: Astrid
authors: A
authors: Gräslund
authors: Free University Berlin, Institute of Experimental Physics, 14195 Berlin, Germany. Electronic address: michael.haumann@fu-berlin.de.
authors: Michael
authors: M
authors: Haumann
mesh_terms: D020816:Amino Acid Motifs; D002692:Chlamydia trachomatis / Q000201:enzymology* / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D007477:Ions; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008027:Light; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry*; D010777:Photochemistry / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013696:Temperature; D014965:X-Rays
chemical_list: D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D008345:Manganese; D007501:Iron; D012264:Ribonucleotide Reductases; D010100:Oxygen
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