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The mechanism and outcome of dioxygen activation by the carboxylate-bridged diiron(II) cluster in the W48F site-directed variant of protein R2 of ribonucleotide reductase from Escherichia coli are determined by kinetic, spectroscopic and chemical methods. The result provides convincing evidence for the rapid and nearly quantitative formation of the Fe(IV)Fe(III) cluster and the Y122 radical species as a consequence of the defect in electron injection in the W48F-substituted variant.

Several rapid kinetic methods were employed to examine the assembly of the essential diferric cluster/tyrosyl radical cofactor of the R2 subunit of Escherichia coli ribonucleotide reductase from apoR2 with Fe2+ and O2 or diferrous R2 with O2. An intermediate X oxidizes tyrosine 122 to the tyrosyl radical concomitant with its own reduction to the diferric cluster generating the R2 cofactor. Various structural models that are consistent with the experimental data are presented.

The outcome of oxygen activation at the Diiron cluster in the protein R2 of ribonucleotide reductase from the F208Y mutant of Escherichia coli was studied by electron injection through a specific pathway. Results indicated that the oxygen activation in the F208Y mutant of R2 partitions between electron outcomes becomes predominant under conditions which favor electron injection. This suggested that surface residue W48F is part of a specific electron-transfer pathway into the R2 cluster site of R2-F208Y.

The two crystalline forms of the six-coordinate complex [Fe-(TpivPP)(NO2)(NO)]- synthesis and characterization were investigated. New (porphinato)-iron(II) complexes having nitric oxide and nitrite were examined to determine their bonding and electronic structure. Iron(II) nitrite derivatives starts the syntheses of {FeNO}7 complexes characterized by a combination of spectroscopic methods and X-ray diffraction studies. Results demonstrate the relative importance of the relative effects of axial ligand orientation in defining electronic structure in iron porphyrins.

Hydropathy analysis indicates three cysteines (C109, C200 and C209) in extracellular loops of the rat serotonin transporter (SERT). These residues, alone or in combination, were mutated to either alanine or serine and the mutant transporters in HeLa cells were expressed utilizing the vaccinia-T7 transient expression system. Findings indicated the possibility that C200 and C209 may be linked by a disulfide bond in the second external loop of SERT.

A reanalysis of Mossbauer spectroscopy using results from earlier studies on the paramagnetic intermediate, X, in the cofactor assembly reaction in Escherichia coli ribonucleotide reductase was conducted for a fuller description of its electronic structure. Results showed isomer shifts of delta(FeA) = 0.56(3) mm/s and delta(Feb) = 0.26(4) mm/s. According to the reduced isomer shift and the hyperfine anisotropy of FeB, X can be best described as a spin-coupled Fe(super III)/Fe(super IV) center without a radical, but with significant spin delocalization onto the oxygen ligand(s).

Evidence for a much longer-lived peroxodiiron(III) intermediate during O2 activation by a site-directed mutant of RNR-R2, in which a single aspartate iron ligand (D84) is altered to glutamate (which is present at the corresponding site in MMOH) is presented. The spectroscopic similarity of the intermediate with H(sub peroxo) indicates that the two have homologous structures. A stable tyrosyl radical is produced by the decay of the intermediate.

The differential iron(II) affinity of the sites of the diiron clusters was investigated through the utilization of two-iron-isotope reaction protocol and Mossbauer spectroscopy. Isotope trapping results have shown that there was a three-fold greater intensity from site 1, indicating that the site 2 was preferentially occupied and site 1 preferentially unoccupied following complexation with low ratios of Fe2+/R2.