Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys₄Fe₂S₂ferredoxin

Abstract

The first reconstitution of an Fe₂S₂ferredoxin with a diamagnetic prosthetic group was recently described[Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625–6626]. Thereplacement of the iron–sulfur cluster of the bacterial ferredoxinputidaredoxin (Pdx) by gallium (Ga³⁺) renders the proteindiamagnetic and permits the use of high-resolution NMR methods to identifyresonances near the metal binding site. We now describe structural featuresof the metal binding site that are not observable by standard NMR methods innative Pdx due to paramagnetic line broadening. These results provide thefirst example of high-resolution NMR-derived structural data concerning themetal binding domain of an Fe₂S₂ ferredoxin, andthe first structural information of any sort for the metal binding site of aferredoxin from this class, which includes adrenodoxin, placental ferredoxinand terpredoxin. Assignments were obtained by applying multidimensional NMRmethods to a series of selectively and nonselectively ¹⁵N- and¹³C/¹⁵N-labeled GaPdx samples. For mostexperiments, a mutant of Pdx was used in which a nonligatingCys⁸⁵ is replaced by serine. All of the major structuralfeatures that were identified in native Pdx are conserved in GaPdx. Theoverall protein dynamics is considerably faster in GaPdx than in the nativeprotein, as reflected by amide proton exchange rates. The C-terminalresidue, Trp¹⁰⁶, also exhibits considerable mobility, asindicated by ¹⁵N{¹H} NOE and ¹⁵NT₁ values of the C-terminal residue of the protein.