Electron paramagnetic resonance measurements of the ferrous mononuclear site of phthalate dioxygenase substituted with alternate divalent metal ions:Direct evidence for ligation of two histidines in the copper(II)-reconstituted protein

The metalloenzyme phthalate dioxygenase (PDO) contains two iron-based sites . A Rieske-type [2Fe-2S] cluster serves as an electron-transferring cofacto r,and a mononuclear iron site is the putative site of substrate oxygenation . A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxin doma in, transfers electrons from NADH to the Rieske center. Any of the metal io ns, Fe(II), Cu(TT), Co(II), Mn(TI), and Zn(II), can be used to populate the mononuclear site, but only Fe(II) is competent for effecting hydroxylation . Nevertheless, studies of how these metal ions affect both the EPR spectra of the reduced Rieske site and the kinetics of electron transfer in the PD O system indicated that each of these metal ions binds tightly and affects the protein similarly. In this study, EPR spectra were obtained from sample s in which iron of the mononuclear site was replaced with Cu(II). The use o f Cu-63(II), in combination with PDO obtained from cultures grown on media enriched in N-15 [using ((NH4)-N-15)(2)SO4 as a sole nitrogen source], [del ta,epsilon-N-15]histidine, as well as natural abundance sources of nitrogen , enabled detailed spectral analysis of the superhyperfine structure of the Cu(II) EPR lines. These studies clearly show that two histidines are coord inated 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 the presence of one or two w ater molecules coordinated to the iron, it is suggested that the mononuclea r site is similar to several other mononuclear nonheme iron proteins, inclu ding naphthalene dioxygenase, for which crystal structures are available. T he lack of observable EPR interaction signals between Cu(II) in the mononuc lear site and the reduced Rieske center of PDO suggest that the two sites a re at least 12 Angstrom apart, which is similar to that found in the naphth alene dioxygenase crystal structure.