ELECTRON-PARAMAGNETIC-RESONANCE STUDY OF THE MIXED-VALENT DIIRON CENTER IN ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE PRODUCED BY REDUCTION OF RADICAL-FREE PROTEIN-R2 AT 77-K

It is demonstrated that gamma-irradiation at 77 K of glycerol-containing solutions of metR2, the radical-free form of protein R2 of ribonucleoside diphosphate reductase (EC 1.17.4.1) from Escherichia coli, or of methemerythrin from Themiste zostericola, gives high yields of mixed-valent (Fe2+, Fe3+) antiferromagnetically coupled states with S = 1/2. These sites are nonequilibrium form with axial electron paramagnetic resonance (EPR) spectra, characterized by g(av) < 2, which are fairly easy to saturate with microwave power. Upon annealing at temperature above 140 K, these primary species are transformed into new mixed-valent species, reflecting relaxation of the ligands of the iron sites toward equilibrium configuration. The mixed-valent nonequlibrium centers of hemerythrin relax at T > 170 K to a form with an EPR spectrum observable only below 35 K. This spectrum is identical to that obtained after chemical reduction of metHr. The antiferromagnetic coupling is maintained, but weakened, and the oxo bridge has been transformed into a hydroxo bridge. In the case of the mixed-valent centers of R2, the relaxation process is more complex. The ligands of the primary nonequilibrium form relax at 142 K into slightly different orientations, reflected in a change of the EPR spectrum from axial into rhombic symmetry. The change of the average g value is small, and the EPR resonance is still easy to saturate. Annealing at 165 K leads to a dramatic change of the EPR spectrum. The absorption at g < 2, corresponding to antiferromagnetically coupled centers, decreases, and new EPR features at low field appear, demonstrating formation of ferromagnetically coupled (Fe2+, Fe3+) centers with S = 9/2. A possible explanation is that a hydroxo or aquo bridge has been formed by protonation of the oxo bridge in the iron center. This EPR spectrum is difficult to saturate by microwaves, and may only be observed at temperatures below ca. 35 K. Annealing at about 200 K causes further changes of the spectral features. It is likely that adjustments of the iron-iron distance and positioning and orientation of ligands are taking place at these temperatures. Annealing at T greater than or equal to 230 K causes disappearance of the low-field EPR spectrum, more rapidly in the presence of O-2 than in its absence. The results show that one-electron reduction of mu-oxo-bridged (Fe3+, Fe3+) centers in proteins at 77 K generates nonequilibrium kinetically stabilized mixed-valent states. These primary products relax to new states upon annealing at T > 77 K. The route and final result of this structural relaxation are different for different proteins.