Active site reactant center geometry in the Co(II)-product radical pair state of coenzyme B12-dependent ethanolamine deaminase determined by using orientation-selection electron spin-echo envelope modulation spectroscopy

The distances and orientations among reactant centers in the active site of coenzyme B12-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized in the Co(II)-product radical pair state by using X-band electron paramagnetic resonance (EPR) and two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopies in the disordered solid state. The unpaired electron spin in the product radical is localized on C2. Our approach is based on the orientation-selection created in the EPR spectrum of the biradical by the axial electron-electron dipolar interaction. Simulation of the EPR line shape yielded a best-fit Co(II)-C2 distance of 9.3 A. ESEEM spectroscopy performed at four magnetic field values addressed the hyperfine coupling of the unpaired electron spin on C2 with 2H in the C5' methyl group of 5'-deoxyadenosine and in the beta-2H position at C1 of the radical. Global ESEEM simulations (over the four magnetic fields) were weighted by the orientation dependence of the EPR line shape. A Nelder-Mead direct search fitting algorithm was used to optimize the simulations. The results lead to a partial model of the active site, in which C5' is located a perpendicular distance of 1.6 A from the Co(II)-C2 axis, at distances of 6.3 and 3.5 A from Co(II) and C2, respectively. The van der Waals contact of the C5'-methyl group and C2 indicates that C5' remains close to the radical species during the rearrangement step. The C2-Hs-C5' angle including the strongly coupled hydrogen, Hs, and the C5'-Hs orientation relative to the C1-C2 axis are consistent with a linear hydrogen atom transfer coordinate and an in-line acceptor p-orbital orientation. The trigonal plane of the C2 atom defines sub-spaces within the active site for C5' radical migration and hydrogen atom transfers (side of the plane facing Co(II)) and amine migration (side of the plane facing away from Co(II)).     

Methionine radical cation: structural studies as a function of pH using X- and Q-band time-resolved electron paramagnetic resonance spectroscopy

A comprehensive high resolution electron paramagnetic resonance (EPR) characterization of the l-methionine radical cation and its N-acetyl derivative in liquid solution at room temperature is presented. The cations were generated photochemically in high yield by excimer laser excitation of a water soluble dye, anthraquinone sulfonate sodium salt, the excited triplet state of which is quenched by electron transfer from the side chain sulfur atom of methionine or N-acetylmethionine. The radicals were detected by continuous wave (CW) time-resolved electron paramagnetic resonance (TREPR) spectroscopy at X-band (9.5 GHz) and Q-band (35 GHz) microwave frequencies. At pH values well below the pK(a) of the protonated amine nitrogen, the cation forms a dimer with another ground-state methionine molecule through a S-S three-electron bond. In basic solution, the lone pair on the nitrogen of the amino acid is available to make an intramolecular S-N three-electron bond with the side chain sulfur atom, leading to a five-membered ring structure for the cation. When the amino acid nitrogen is unsubstituted (methionine itself), rapid deprotonation to an aminyl radical takes place at high pH values. If the nitrogen is substituted (N-acetylmethionine), the cyclic structure is observed within its electron spin relaxation time at about 1 micros. Spectral simulation provides chemical shifts (g-factors) and hyperfine coupling constants for all structures, and isotopic labeling experiments strongly support the assignments.     

Assessment of the existence of hyper-long axial Co(II)-N bonds in cobinamide B(12) models by using electron paramagnetic resonance spectroscopy.

Protein control of cobalt-axial nitrogen ligand bond length has been proposed to modulate the reactivity of vitamin B(12) coenzyme during the catalytic cycle of B(12)-dependent enzymes. In particular, hyper-long Co-N bonds may favor homolytic cleavage of the trans-cobalt-carbon bond in the coenzyme. X-ray crystallographic studies point to hyper-long bonds in two B(12) holoenzymes; however, mixed redox and ligand states in the crystals thwart clear conclusions. Since EPR theory predicts an increase in Co(II) hyperfine splitting as donation from the axial N-donor ligand decreases, EPR spectroscopy could clarify the X-ray results. However, the theory is apparently undermined by the similar splitting reported for the 2-picoline (2-pic) and pyridine (py) adducts of Co(II) cobinamide (Co(II)Cbi(+)), adducts thought to have long and normal Co-N axial bond lengths, respectively. Cobinamides, with the B(12) 5,6-dimethylbenzimidazole loop removed, are excellent B(12) models. We studied Co(II)Cbi(+) adducts of unhindered 4-substituted pyridines (4-X-py's) in ethylene glycol to separate orbital size effects from Co-N axial distance effects on these splittings. The linear increase in splitting with the decrease in 4-X-py basicity found is consistent with the theoretically predicted increase in unpaired electron spin density as axial N lone pair donation to Co(II) decreases. No adduct (and hence no hyper-long Co(II)-N axial bond) was formed even by 8 M 2-pic, if the 2-pic was purified by a novel Co(III)-affinity distillation procedure designed to remove trace nitrogenous ligand impurities present in 2-pic distilled in the regular manner. Adducts formed by impurities in 2-pic and other hindered pyridines misled previous investigators into attributing results to adducts with long Co-N bonds. We find that many 2-substituted py's known to form adducts with simple synthetic Co models do not bind Co(II)Cbi(+). Thus, the equatorial corrin ring sterically impedes binding, making Co(II)Cbi(+) a highly selective binding agent for unhindered sp(2) N-donor ligands. Our results resolve the apparent conflict between EPR experiment and theory. The reported Co(II) hyperfine splitting of the enzyme-bound cofactor in five B(12) enzymes is similar to that of the relevant free cofactor. The most reasonable interpretation of this similarity is that the Co-N axial bond of the bound cofactor is not hyper-long in any of the five cases.     

Generation and characterization of the selenocysteinyl radical: direct evidence from time-resolved UV/Vis, electron paramagnetic resonance, and Fourier transform infrared spectroscopy

The selenocysteinyl radical 1 has been generated for the first time by laser flash photolysis (lambda(exc) = 266 nm) of dimethyl bis(N-tert-butoxycarbonyl)-l-selenocystine 2 and of [(9-fluorenylideneamino)oxycarbonyl]methyl(N-tert-butoxycarbonyl)-l-selenocysteine 3 in acetonitrile and characterized by time-resolved (TR) UV/Vis, Fourier transform infrared (FTIR), and electron paramagnetic spectroscopy in combination with theoretical methods. A detailed product study was conducted using gas chromatography and one- and two-dimensional NMR spectroscopy. In the case of [(9-fluorenylideneamino)oxycarbonyl]methyl(N-tert-butoxycarbonyl)-l-selenocysteine 3, the (9-fluorenylideneamino)oxycarbonyl moiety serves as a photolabile protection group providing a "caged selenocysteinyl radical" suitable for biophysical applications. Cleavage of the diselenide bridge or the selenium-carbonyl bond by irradiation is possible in high quantum yields. Because of the lack of a good IR chromophore in the mid-IR region, the selenocysteinyl radical 1 cannot be monitored directly by TR FTIR spectroscopy. TR UV/Vis spectroscopy revealed the formation of the selenocysteinyl radical 1 from both precursors. The selenocysteinyl radical 1 has a lifetime tau approximately 63 mus and exhibits a strong band located at lambda(max) = 335 nm. Calculated UV absorptions of the selenocysteinyl radical (UB3LYP/6-311G(d,p)) are in good agreement with the experimental results. The use of TR UV/Vis spectroscopy permitted the determination of the decay rates of the selenocysteinyl radical in the presence of two quenchers. The product studies demonstrated the reversible photoreaction of dimethyl bis(N-tert-butoxycarbonyl)-l-selenocystine 2. Products of the photolysis of the "caged selenocysteinyl radical" precursor 3 are dimethyl bis(N-tert-butoxycarbonyl)-l-selenocystine 2, carbon dioxide, and some further smaller fragments. In addition, the photodecomposition of the (9-fluorenylideneamino)oxycarbonyl moiety produced 9-fluorenone-oxime 4, 9-fluoren-imine 5, and 6 and 7 as products of the dimerization of two 9-fluorenoneiminoxy radicals 8.     

Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: observation of the B-side radical pair P700(+)A1B(-) in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700(+)A1A(-) and P700(+)A1B(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700(+)A1A(-). A detailed comparison reveals different orientations of A1A(-) and A1B(-) in their respective binding sites such that formation of a strong hydrogen bond from A1(-) to the protein backbone is possible only in the case of A1A(-). We suggest that this is relevant to the rates of forward electron transfer from A1A(-) or A1B(-) to the iron-sulfur center F(X), which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.