Hemes are among the most prevalent and versatile cofactors in biological systems. One of the factors affecting the adaptability of these cofactors to a wide variety of different biological needs is the ease with which the redox state, and within each redox state, the electronic state of the iron, may be adjusted by porphyrin substituents and by protein environment. During the next grant period the investigator will concentrate on three projects: I. The effect(s) of axial ligands, heme substituents and heme ring reduction state on the electronic ground state of low-spin Fe(III) highly ruffled porphyrins and hydroporphyrins will be investigated utilizing a combination of NMR, EPR, ESEEM, Mossbauer and near-IR MCD spectroscopies and x-ray crystallography. Complexes with strong pi-acceptor ligands will be used to push toward the interface between the (dxy)1 and (a2u)1 electronic ground states. Spin-admixed perchloratoiron(III) complexes of 'hindered' porph-yrinates and reduced hemes will also be investigated as a function of temperature in order to determine the effect of macrocycle substituents on the nature of the electronic -ground state of these models of the cytochromes c'. II. A series of new low-spin Fe(III) and Fe(II) porphyrinates with appended bulky Mo(V) centers will be investigated in order to determine the importance of parallel vs. perpendicular axial ligand plane orientation on the reduction potentials of model hemes and heme proteins. ESEEM studies of selected model hemes and heme proteins will be carried out in order to define the orientations of the magnetic axes with respect to the nodal planes of the axial ligands. 1- and 2D 1H and 13C NMR studies of chelated protohemin cyanide complexes, some of which will be isotopically labelled with 13C by biosynthetic techniques developed in this laboratory, will be carried out to completely assign the resonances of the heme ring for the two (inseparable) isomers both in homogeneous solution and in the frozen (glassy) state; studies of the NMR spectra of the isotopically labelled protohemins reconstituted into myoglobin will also be carried out for comparison. 57Fe NMR studies of enriched chelated protoheme isomers with PMe as axial ligand will be used to determine the effect of a fixed ligand plane on the 31P-57Fe coupling constant; these studies will then be extended to the PMe3, complexes of myoglobin and to cytochrome b5. III. Several proteins in which a heme axial ligand has been displaced, or replaced by site-directed mutagenesis with a residue with a small side chain, will be prepared and their properties in the presence of exogenous ligands that will alter the orientation of axial ligand planes will be investigated by all above-mentioned techniques.
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