Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra.

Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E0. Two of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.

The Radical SAM Heme Synthase AhbD from Methanosarcina barkeri Contains Two Auxiliary [4Fe-4S] Clusters.

In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron-sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary [4Fe-4S] cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron-sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary [4Fe-4S] clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer.

A Cobalamin-Dependent Radical SAM Enzyme Catalyzes the Unique Cα -Methylation of Glutamine in Methyl-Coenzyme†M Reductase.

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 Cα -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 sp3 -hybridized carbon atoms.

Direct EPR detection of atomic nitrogen in an atmospheric nitrogen plasma jet.

In this study, an atmospheric nitrogen plasma jet generated by a custom-built micro-plasma device was analyzed at room temperature by continuous wave and pulse EPR spectroscopy in real time. Transiently formed nitrogen atoms were detected without the necessity to use spin-traps or other reagents for their stabilization. In contrast to results from optical emission spectroscopy, only signals from the 4S ground state of 14N and 15N could be detected. EPR data analysis revealed an isotropic g value of 1.9971 and isotropic hyperfine coupling constants of a(14N) = (10.47 ± 0.02) MHz and a(15N) = (14.69 ± 0.02) MHz. Moreover, lifetime and relaxation data could be determined; both are discussed in terms of spectral widths and actual concentrations of the transiently formed nitrogen species within the plasma jet. The data show that the lifetimes of atomic nitrogen and charged particles such as N+ must be different, and for the latter below the observation time window of EPR spectroscopy. We demonstrate that the real-time (pulsed) EPR technique is a fast and reliable alternative to detect atomic nitrogen in atmospheric pressure plasma jets. The method may be used for a continuous monitoring of the quality of plasma jets.

Coupled Methyl Group Rotation in FMN Radicals Revealed by Selective Deuterium Labeling.

Flavin semiquinones are common intermediate redox states in flavoproteins, and thus, knowledge of their electronic structure is essential for fully understanding their chemistry and chemical versatility. In this contribution, we use a combination of high-field electron nuclear double resonance spectroscopy and selective deuterium labeling of flavin mononucleotide (FMN) with subsequent incorporation as cofactor into a variant Avena sativa LOV domain to extract missing traits of the electronic structure of a protein-bound FMN radical. From these experiments, precise values of small proton hyperfine and deuterium nuclear quadrupole couplings could be extracted. Specifically, isotropic hyperfine couplings of -3.34, -0.11, and +0.91 MHz were obtained for the protons H(6), H(9), and H(7α), respectively. These values are discussed in the light of specific protein-cofactor interactions. Furthermore, the temperature behavior of the H(7α) methyl-group rotation elicited by its energy landscape was analyzed in greater detail. Pronounced interplay between the two methyl groups at C(7) and C(8) of FMN could be revealed. Most strikingly, this rotational behavior could be modulated by selective deuterium editing.

Long-Lived Hydrated FMN Radicals: EPR Characterization and Implications for Catalytic Variability in Flavoproteins.

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.

Direct observation of a deoxyadenosyl radical in an active enzyme environment.

5'-deoxyadenosyl radicals have been proposed as the first common intermediate in the molecular reaction mechanism of the family of radical S-adenosyl-l-methionine (SAM) enzymes. However, this radical species has not yet been directly observed in a catalytically active enzyme environment. In a reduced and SAM-containing C140A mutant of the spore photoproduct lyase from Geobacillus thermodenitrificans, a mutant with altered catalytic activity, we were able to identify an organic radical with pronounced hyperfine structure using electron paramagnetic resonance spectroscopy. Guided by quantum-chemical computations at the density functional theory level of theory, this radical could be tentatively assigned to a deoxyadenosyl radical, which provides first experimental evidence for this intermediate in the reaction mechanism of radical SAM enzymes.

Cationic cluster formation versus disproportionation of low-valent indium and gallium complexes of 2,2'-bipyridine.

Group 13 M(I) compounds often disproportionate into M(0) and M(III). Here, however, we show that the reaction of the M(I) salt of the weakly coordinating alkoxyaluminate [Ga(I)(C6H5F)2](+)[Al(OR(F))4](-) (R(F)=C(CF3)3) with 2,2'-bipyridine (bipy) yields the paramagnetic and distorted octahedral [Ga(bipy)3](2+)(•){[Al(OR(F))4](-)}2 complex salt. While the latter appears to be a Ga(II) compound, both, EPR and DFT investigations assign a ligand-centred [Ga(III){(bipy)3}(•)](2+) radical dication. Surprisingly, the application of the heavier homologue [(I)n(I)(C6H5F)2](+)[Al(OR(F))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(OR(F))4](-)}3 and [M4](4+){[Al(OR(F))4](-)}4 salts.

Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome - protonated and nonprotonated flavin radical-states.

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(•-) 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.