The Internal Dynamics of Mini c TAR DNA Probed by Electron Paramagnetic Resonance of Nitroxide Spin-Labels at the Lower Stem, the Loop, and the Bulge

Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2′-aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ₃ RNA, but at 5 °C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3′ and 5′ termini showed evidence of an NCp7-induced increase in the 3′–5′ end-to-end distance distribution and a partially melted stem

EPR–ENDOR Characterization of (¹⁷O, ¹H, ²H) Water in Manganese Catalase and Its Relevance to the Oxygen-Evolving Complex of Photosystem II

The synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (OEC) of photosystem II (PSII). Understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the OEC. Mn catalase in its Mn­(III)–Mn­(IV) state is a protein model of the OEC’s S₂ state. From ¹⁷O-labeled water exchanged into the di-μ-oxo di-Mn­(III,IV) coordination sphere of Mn catalase, CW Q-band ENDOR spectroscopy revealed two distinctly different ¹⁷O signals incorporated in distinctly different time regimes. First, a signal appearing after 2 h of ¹⁷O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the ¹⁷O μ-oxo hyperfine coupling of the di-μ-oxo di-Mn­(III,IV) bipyridine model (Usov, O. M.; Grigoryants, V. M.; Tagore, R.; Brudvig, G. W.; Scholes, C. P. J. Am. Chem. Soc. 2007, 129, 11886−11887), this signal was assigned to μ-oxo oxygen. EPR line broadening was obvious from this ¹⁷O μ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-μ-oxo position, from which ¹⁷O ENDOR showed a smaller 3.8 MHz hyperfine coupling and possible quadrupole splittings, indicating a terminal water of Mn­(III). Exchangeable proton/deuteron hyperfine couplings, consistent with terminal water ligation to Mn­(III), also appeared. Q-band CW ENDOR from the S₂ state of the OEC was obtained following multihour ¹⁷O exchange, which showed a ¹⁷O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as μ-oxo-¹⁷O by resemblance to the μ-oxo signals from Mn catalase and the di-μ-oxo di-Mn­(III,IV) bipyridine model