The solar water-splitting protein complex, photosystem II, catalyzes the light-driven oxidation of water to dioxygen in Nature. The four-electron oxidation reaction of water occurs at the tetranuclear manganese-calcium-oxo catalytic cluster that is present in the oxygen-evolving complex of photosystem II. The mechanism of light-driven water oxidation has been a subject of intense interest, and the oxygen-evolving complex of photosystem II has been studied extensively by structural and biochemical methods. While the recent X-ray crystal structures and single-crystal EXAFS investigations provide a model for the geometry of the tetranuclear manganese-calcium-oxo catalytic cluster, there is limited knowledge of the protein environment that surrounds the catalytic cluster. In this study, we demonstrate the application of two-dimensional hyperfine sublevel correlation spectroscopy to determine the magnetic couplings of the catalytic cluster with the (14)N atoms of surrounding amino acid residues in the S(2) state of the oxygen-evolving complex of photosystem II. We utilize two-dimensional difference spectroscopy to facilitate unambiguous assignments of the spectral features and identify at least three separate (14)N atoms that are interacting with the catalytic cluster in the S(2) state. The results presented here, for the first time, identify previously unknown ligands to the catalytic cluster of photosystem II and provide avenues for the assignment of residues by site-directed mutagenesis and the refinement of computational and mechanistic models of photosystem II.

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Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Installation of olefins into molecules is a key transformation in organic synthesis. The recently discovered decarboxylation-assisted olefination in the biosynthesis of rhabduscin by a mononuclear non-heme iron enzyme (P.IsnB) represents a novel approach in olefin construction. This method is commonly employed in natural product biosynthesis. Herein, we demonstrate that a ferryl intermediate is used for C–H activation at the benzylic position of the substrate. We further establish that P.IsnB reactivity can be switched from olefination to hydroxylation using electron-withdrawing groups appended on the phenyl moiety of the analogues. These experimental observations imply that a pathway involving an initial C–H activation followed by a benzylic carbocation species or by electron transfer coupled β- scission is likely utilized to complete C=C bond formation.

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