The role of arginine-127 at the proximal NO-binding site in determining the electronic structure and function of 5-coordinate NO-heme in cytochrome c' of Rhodobacter sphaeroides.

Cytochrome c' is a heme protein from a denitrifying variant of Rhodobacter sphaeroides which may serve to store and transport metabolic NO while protecting against NO toxicity. Its heme site bears resemblance through its 5-coordinate NO-binding capability to the regulatory site in soluble guanylate cyclase. A conserved arginine (Arg-127) abuts the 5-coordinate NO-heme binding site, and the alanine mutant R127A provided insight into the role of the Arg-127 in establishing the electronic structure of the heme-NO complex and in modifying the heme-centered redox potential and NO-binding affinity. By comparison to R127A, the wild-type Arg-127 was determined to increase the heme redox potential, diminish the NO-binding affinity, perturb and diminish the 14NO hyperfine coupling determined by ENDOR (electron nuclear double resonance), and increase the maximal electronic g-value. The larger isotropic NO hyperfine and the smaller maximal g-value of the R127A mutant together predicted that the Fe-N-O bond angle in the mutant is larger than that of the Arg-127-containing wild-type protein. Deuterium ENDOR provided evidence for exchangeable H/D consistent with hydrogen bonding of Arg-127, but not Ala-127, to the O of the NO. Proton ENDOR features previously assigned to Phe-14 on the distal side of the heme were unperturbed by the proximal side R127A mutation, implying the localized nature of that mutational perturbation at the proximal, NO-binding side of the heme. From this work two functions of positively charged Arg-127 emerged: the first was to maintain the KD of the cytochrome c' in the 1 microM range, and the second was to provide a redox potential that enhances the stability of the ferrous heme

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 S(2) state. From (17)O-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 (17)O signals incorporated in distinctly different time regimes. First, a signal appearing after 2 h of (17)O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the (17)O 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 (17)O μ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-μ-oxo position, from which (17)O 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 S(2) state of the OEC was obtained following multihour (17)O exchange, which showed a (17)O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as μ-oxo-(17)O by resemblance to the μ-oxo signals from Mn catalase and the di-μ-oxo di-Mn(III,IV) bipyridine model

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 ψ(3) 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

Porphyrin–phospholipid liposomes permeabilized by near-infrared light

The delivery of therapeutic compounds to target tissues is a central challenge in treating disease. Externally controlled drug release systems hold potential to selectively enhance localized delivery. Here we describe liposomes doped with porphyrin–phospholipid that are permeabilized directly by near-infrared light. Molecular dynamics simulations identified a novel light-absorbing monomer esterified from clinically approved components predicted and experimentally demonstrated to give rise to a more stable porphyrin bilayer. Light-induced membrane permeabilization is enabled with liposomal inclusion of 10 molar % porphyrin–phospholipid and occurs in the absence of bulk or nanoscale heating. Liposomes reseal following laser exposure and permeability is modulated by varying porphyrin–phospholipid doping, irradiation intensity or irradiation duration. Porphyrin–phospholipid liposomes demonstrate spatial control of release of entrapped gentamicin and temporal control of release of entrapped fluorophores following intratumoral injection. Following systemic administration, laser irradiation enhances deposition of actively loaded doxorubicin in mouse xenografts, enabling an effective single-treatment antitumour therapy