The metalloenzyme phthalate dioxygenase (PDO) contains two iron-based sites. A Rieske-type [2Fe-2S] cluster serves as an electron-transferring cofactor, and a mononuclear iron site is the putativesite of substrate oxygenation. A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxindomain, transfers electrons from NADH to the Rieske center. Any of the metal ions, Fe(II), Cu(II), Co(II),Mn(II), and Zn(II), can be used to populate the mononuclear site, but only Fe(II) is competent for effectinghydroxylation. Nevertheless, studies of how these metal ions affect both the EPR spectra of the reducedRieske site and the kinetics of electron transfer in the PDO system indicated that each of these metal ionsbinds tightly and affects the protein similarly. In this study, EPR spectra were obtained from samples inwhich iron of the mononuclear site was replaced with Cu(II). The use of
63Cu(II), in combination withPDO obtained from cultures grown on media enriched in
15N [using (
15NH
4)
2SO
4 as a sole nitrogen source],[
,
-
15N]histidine, as well as natural abundance sources of nitrogen, enabled detailed spectral analysis ofthe superhyperfine structure of the Cu(II) EPR lines. These studies clearly show that two histidines arecoordinated to the mononuclear site. Coupled with previous studies [
Bertini, I., Luchinat, C., Mincione,G., Parigi, G., Gassner G. T., and Ballou, D. P. (1996)
J. Bioinorg. Chem. 1, 468-475] that show thepresence of one or two water molecules coordinated to the iron, it is suggested that the mononuclear siteis similar to several other mononuclear nonheme iron proteins, including naphthalene dioxygenase, forwhich crystal structures are available. The lack of observable EPR interaction signals between Cu(II) inthe mononuclear site and the reduced Rieske center of PDO suggest that the two sites are at least 12 Åapart, which is similar to that found in the naphthalene dioxygenase crystal structure.