\u3csup\u3e15\u3c/sup\u3eN SOLID-STATE NMR DETECTION OF FLAVIN PERTURBATION BY H-BONDING IN MODELS AND ENZYME ACTIVE SITES

Massey and Hemmerich proposed that the different reactivities displayed by different flavoenzymes could be achieved as a result of dominance of different flavin ring resonance structures in different binding sites. Thus, the FMN cofactor would engage in different reactions when it had different electronic structures. To test this proposal and understand how different protein sites could produce different flavin electronic structures, we are developing solid-state NMR as a means of characterizing the electronic state of the flavin ring, via the 15N chemical shift tensors of the ring N atoms. These provide information on the frontier orbitals. We propose that the 15N chemical shift tensors of flavins engaged in different hydrogen bonds will differ from one another. Tetraphenylacetyl riboflavin (TPARF) is soluble in benzene to over 250 mM, so, this flavin alone and in complexes with binding partners provides a system for studying the effects of formation of specific hydrogen bonds. For N5, the redoxactive N atom, one of the chemical shift principle values (CSPVs) changed 10 ppm upon formation of a hydrogen bonded complex, and the results could be replicated computationally. Thus our DFT-derived frontier orbitals are validated by spectroscopy and can be used to understand reactivity. Indeed, our calculations indicate that the electron density in the diazabutadiene system diminishes upon H-bond complex formation, consistent with the observed 100 mV increase in reduction midpoint potential. Thus, the current studies of TPARF and its complexes provide a useful baseline for further SSNMR studies aimed at understanding flavin reactivity in enzymes

Calculating the NMR Chemical Shielding of Large Molecules

This thesis examines three approximations that significantly reduce the computational time of theoretical NMR shielding calculations for large molecules, whilst largely retaining the accuracy of the parent method: fragmentation, locally dense basis sets and composite methods. For fragmentation it is established that Level 4 fragments reliably reproduce full molecule shieldings, when hydrogen bonds are treated as single bonds, and long range through space corrections are incorporated through the McConnell equation and background charges. The pcS-n basis set family is demonstrated to converge more rapidly towards the basis set limit than all other examined families. Furthermore, it is established that this limit is consistent with convergence towards experimental values. A systematic investigation of locally dense basis sets established that a group based partitioning of the pcS-4, pcS-2 and pcS-1 basis sets, augmented with through space allocations, allowed the shielding to be produced within chemical accuracy for a variety of compounds. Finally, composite methods utilising a variety of levels of theory were systematically investigated, and it was found that a double composite method combining the HF, MP2 and CCSD(T) levels of theory and the pcS-4, pcS-2 and pcS-1 basis sets yielded NMR shieldings that were within chemical accuracy of CCSD(T)/pcS-4 calculations, themselves having converged closely to experimental values. When considered in combination this work represents a significant step towards achieving chemical accuracy for protein NMR shielding calculations