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 cofactor, and a mononuclear iron site is the putative site of substrate oxygenation. A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxin domain, 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 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 PDO system indicated that each of these metal ions binds tightly and affects the protein similarly. In this study, EPR spectra were obtained from samples in which iron of the mononuclear site was replaced with Cu(II). The use of (63)Cu(II), in combination with PDO obtained from cultures grown on media enriched in (15)N [using ((15)NH(4))(2)SO(4) as a sole nitrogen source], [delta,epsilon-(15)N]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 coordinated 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 water molecules coordinated to the iron, it is suggested that the mononuclear site is similar to several other mononuclear nonheme iron proteins, including naphthalene dioxygenase, for which crystal structures are available. The lack of observable EPR interaction signals between Cu(II) in the mononuclear site and the reduced Rieske center of PDO suggest that the two sites are at least 12 A apart, which is similar to that found in the naphthalene dioxygenase crystal structure.
Coulter
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ED
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Moon
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N
Batie
C J
CJ
Dunham
W R
WR
Ballou
D P
DP
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D016956:Burkholderia cepacia / Q000201:enzymology; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry
D001051:Apoenzymes; D002413:Cations, Divalent; D005290:Ferric Compounds; D005296:Ferrous Compounds; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; C037989:Rieske iron-sulfur protein; D014443:Tyrosine; D006639:Histidine; D003300:Copper; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase; C044732:phthalate dioxygenase; D014450:Electron Transport Complex III