Drosophila melanogaster cryptochrome is one of the model proteins for animal blue-light photoreceptors. Using time-resolved and steady-state optical spectroscopy, we studied the mechanism of light-induced radical-pair formation and decay, and the photoreduction of the FAD cofactor. Exact kinetics on a microsecond to minutes timescale could be extracted for the wild-type protein using global analysis. The wild-type exhibits a fast photoreduction reaction from the oxidized FAD to the FAD•À state with a very positive midpoint potential of~+125 mV, although no further reduction could be observed. We could also demonstrate that the terminal tryptophan of the conserved triad, W342, is directly involved in electron transfer; however, photoreduction could not be completely inhibited in a W342F mutant. The investigation of another mutation close to the FAD cofactor, C416N, rather unexpectedly reveals accumulation of a protonated flavin radical on a timescale of several seconds. The obtained data are critically discussed with the ones obtained from another protein, Escherichia coli photolyase, and we conclude that the amino acid opposite N(5) of the isoalloxazine moiety of FAD is able to (de)stabilize the protonated FAD radical but not to significantly modulate the kinetics of any light-inducted reactions.

Group 13 MI compounds often disproportionate into M0 and MIII. Here, however, we show that the reaction of the MI salt of the weakly coordinating alkoxyaluminate [GaI(C6H5F)2]+[Al(ORF)4]− (RF=C(CF3)3) with 2,2'-bipyridine (bipy) yields the paramagnetic and distorted octahedral [Ga(bipy)3]2+•{[Al(ORF)4]−}2 complex salt. While the latter appears to be a GaII compound, both, EPR and DFT investigations assign a ligand-centred [GaIII{(bipy)3}•]2+ radical dication. Surprisingly, the application of the heavier homologue [InI(C6H5F)2]+[Al(ORF)4]− leads to aggregation and formation of the homonuclear cationic triangular and rhombic [In3(bipy)6]3+, [In3(bipy)5]3+ and [In4(bipy)6]4+ metal atom clusters. Typically, such clusters are formed under strongly reductive conditions. Analysing the unexpected redox-neutral cationic cluster formation, DFT studies suggest a stepwise formation of the clusters, possibly via their triplet state and further investigations attribute the overall driving force of the reactions to the strong In−In bonds and the high lattice enthalpies of the resultant ligand stabilized [M3]3+{[Al(ORF)4]−}3 and [M4]4+{[Al(ORF)4]−}4 salts.

Methyl-coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2-(S)methylglutamine. The enzyme responsible for the C α -methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin-dependent radical SAM enzyme as the glutamine C-methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base-off, His-off conformation and contains a single [4Fe-4S] cluster. The cobalamin cofactor cycles between the methyl-cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5'-deoxyadenosine and S-adenosyl-L-homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C-methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp 3 -hybridized carbon atoms.

Until now, FMN/FAD radicals could not be stabilized in aqueous solution or other protic solvents because of rapid and efficient dismutation reactions. In this contribution, a novel system for stabilizing flavin radicals in aqueous solution is reported. Subsequent to trapping FMN in an agarose matrix, light-generated FMN radicals could be produced that were stable for days even under aerobic conditions, and their concentrations were high enough for extensive EPR characterization. All large hyperfine couplings could be extracted by using a combination of continuous-wave EPR and low-temperature ENDOR spectroscopy. To map differences in the electronic structure of flavin radicals, two exemplary proton hyperfine couplings were compared with published values from various neutral and anionic flavoprotein radicals: C(6)­H and C­(8α)­H 3 . It turned out that FMN•– in an aqueous environment shows the largest hyperfine couplings, whereas for FMNH• under similar conditions, hyperfine couplings are at the lower end and the values of both vary by up to 30%. This finding demonstrates that protein–cofactor interactions in neutral and anionic flavoprotein radicals can alter their electron spin density in different directions. With this aqueous system that allows the characterization of flavin radicals without protein interactions and that can be extended by using selective isotope labeling, a powerful tool is now at hand to quantify interactions in flavin radicals that modulate the reactivity in different flavoproteins.

We describe a novel stacked split-ring type microwave (MW) resonator that is integrated into a 10mm by 10mm sized microfluidic chip. A straightforward and scalable batch fabrication process renders the chip suitable for single-use applications. The resonator volume can be conveniently loaded with liquid sample via microfluidic channels patterned into the mid layer of the chip. The proposed MW resonator offers an alternative solution for compact in-field measurements, such as low-field magnetic resonance (MR) experiments requiring convenient sample exchange. A microstrip line was used to inductively couple MWs into the resonator. We characterised the proposed resonator topology by electromagnetic (EM) field simulations, a field perturbation method, as well as by return loss measurements. Electron paramagnetic resonance (EPR) spectra at X-band frequencies were recorded, revealing an electron-spin sensitivity of 3.7·10(11)spins·Hz(-1/2)G(-1) for a single EPR transition. Preliminary time-resolved EPR experiments on light-induced triplet states in pentacene were performed to estimate the MW conversion efficiency of the resonator.