Mössbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli. A new iron-to-protein stoichiometry.
57Fe-enriched ribonucleotide reductase subunit B2 from Escherichia coli strain N6405/pSPS2 has been characterized by Mössbauer and EPR spectroscopy in its native diferric state and in a new differous form. The native protein exhibits two Mössbauer doublets in a 1:1 ratio with parameters that are in excellent agreement with those reported for the wild-type protein (Atkin, C. L., Thelander, L., Reichard, P., and Lang, G. (1983) J. Biol. Chem. 248, 7464-7472); in addition, our studies show the absence of adventitiously bound iron. The iron content in the present samples approached 4 per B2 subunit, and the tyrosyl radical content exceeded 1 per B2 subunit. The higher values are attributed to the use of a new epsilon 280 for the protein and more efficient methods for iron extraction. We thus propose that subunit B2 has two binuclear iron clusters, each associated with its own tyrosyl radical, in contradistinction from the prevailing model. Reduction of the native protein with dithionite or reconstitution of the apoprotein with Fe(II) afforded a protein complex with Mössbauer parameters, delta EQ = 3.13 mm/s and delta = 1.26 mm/s at 4.2 K, and a low field EPR signal associated with an integer spin system. These spectral properties resemble those of methane monooxygenase in its diferrous form. Upon exposure to O2, the reduced subunit B2 readily converts to the diferric state and yields active enzyme.
Lynch
J B
JB
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Juarez-Garcia
C
C
Münck
E
E
Que
L
L
D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007504:Iron Radioisotopes; D008970:Molecular Weight; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer; D014443:Tyrosine / Q000378:metabolism
D005290:Ferric Compounds; D005296:Ferrous Compounds; D007504:Iron Radioisotopes; D004227:Dithionite; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases