Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O-2-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 O-2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.