The number of iron atoms in the paramagnetic center (G =1.94) of reduced putidaredoxin, a nonheme iron protein.

A number of oxidation-reduction proteins, containing iron and an acid-labile form of sulfide, have been shown to exhibit low-temperature electron paramagnetic resonance (EPR) signals near g = 1.94, on reduction.l-4 These proteins include oxidases of the xanthine type, ferredoxins, and nonheme-iron and iron flavoproteins from mitochondria and bacteria. By substitution with iron5 and sulfur6' I isotopes having nonzero nuclear spin, both elements were conclusively shown to participate in the paramagnetic center. The nuclear magnetic moments may couple to the moment of the unpaired electron of the center, producing small positive and negative incremental local fields which act to split or broaden the EPR spectrum compared to the unsubstituted cases. The effect depends, in the simplest case of isotropic hyperfine interaction, on three variables, viz., the enrichment in isotope of nonzero nuclear spin (Fe57 of SI' in the present case), the effective local field contribution or hyperfine splitting constant of each atom, and the number of atoms of each isotope involved with a single unpaired electron. If the enrichment can be estimated, trial spectra can be calculated for various hyperfine splittings and numbers of atoms. In favorable cases comparison to the observed spectrum will reveal the values of the variables that best fit hypothesis to experiment. In this way one can determine the number and kinds of atoms interacting with the unpaired electron under the assumption of predominantly isotropic hyperfine interaction. This approach has been adopted in the present work where the exchange of Fe57 for Fe`6 (and SI' for S32) in the iron protein, putidaredoxin, of a methylene hydroxylase of Pseudomonas putida8 has allowed the study of the question of how many iron (and sulfur) atoms are involved in the paramagnetic center of this material. In addition, supporting evidence was obtained from an anaerobic reductive titration of the protein. Because of the ubiquitous distribution of the paramagnetic species exhibiting the g = 1.94 signal on reduction as an electron carrier,2' 3 and because this type of EPR signal is atypical of iron compounds with well-understood structures, much discussion of this entity has taken place. Ultimately, the chemical structures and molecular orbital descriptions of these complexes must account for the optical spectra,'3 the redox potentials ranging from +220 to -430 mv,13,8 and the EPR spectra on reduction which feature an average g value less than the free-electron value.2 (Occasionally this last point has not been appreciated, and model complexes'0 with EPR spectra straddling g = 2, in the fashion of low-spin heme complexes, are not pertinent to the g = 1.94 problem. Average g-values are calculated from g2a0 = '/3 (92 + g2 + g2) = '/3 (2g l+ g2)