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

Abstract

Background: Typical FeFe and MnFe cofactors bind to numerous enzymes such as ribonucleotide reductases. Crystallographic data suggest x-ray photoreduction (XPR) effects. Results: Rapid XPR-induced cofactor changes were monitored using time-resolved x-ray absorption spectroscopy. Conclusion: The XPR-induced cofactor states differ significantly from the native configurations, but comply with crystallographic structures. Significance: Structure determination for high-valent dimetal-oxygen cofactors requires free electron-laser protein crystallography combined with x-ray spectroscopy.

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

“…These findings imply that the reduction of the metal center by x-rays does not go through the catalytic pathway. This is similar to the situation found in the case of Fe/Fe and Fe/Mn ribonucleotide reductase, where the states generated by x-ray photoreduction are significantly different from the native catalytic state of the metal center (38).…”

Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor

“…Similarly rapid xpr has been observed for typical R2 proteins (55,56). The faster reduction of Mn III compared with Fe III can likely be explained by the about twice as large x-ray absorption crosssection of the protein sample at manganese K-edge energies compared with iron K-edge energies (56). We used a spot size of about 0.3 mm 2 at ϳ10 11 photons s Ϫ1 , i.e.…”

“…However, this factor may be compensated by the about 10 times lower absorption of the sample at the higher x-ray energies used in crystallography. Assuming a linear dose-rate relationship of xpr (56,59), at least about 200 -10,000 times faster reduction of metal(III) ions was therefore expected during crystallographic data collection of R2lox ox . This means that the Fe II and Mn II levels likely were reached within less than 0.5-25 s of data collection, long before a complete dataset was obtained.…”

“…In oxidized R2lox, metal-metal distances of ϳ3 Å may also correspond to (minor amounts of) R2-like centers formed in the absence of the fatty acid, which contain two metal-bridging O(H) groups. (An ϳ3-Å metal distance is typical for R2 proteins containing M III 2 clusters (55,56).) An in-depth characterization of such minor cofactor species, however, would require samples in which they are quantitatively populated.…”

The Mn 4 Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser

“…Thus, even at cryogenic temperatures, SR-based XRD of the Mn 4 CaO 5 structure in PSII is fundamentally limited by the radiation damage to the redox-active metal site, making it difficult to obtain intact structures of stable or transient reaction intermediates. Not only is this radiation damage a problem for PSII, but it also presents a challenge encountered in many other redox-active metalloenzymes [15][16][17][18]. Thus far, the geometric and electronic structural information of the intact Mn 4 CaO 5 cluster has been primarily addressed by spectroscopic methods [19][20][21][22], especially X-ray absorption (XAS) and emission spectroscopy (XES) techniques [14,[23][24][25].…”