Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys4Fe2S2 ferredoxin.
The first reconstitution of an Fe2S2 ferredoxin with a diamagnetic prosthetic group was recently described [Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625-6626]. The replacement of the iron-sulfur cluster of the bacterial ferredoxin putidaredoxin (Pdx) by gallium (Ga3+) renders the protein diamagnetic and permits the use of high-resolution NMR methods to identify resonances near the metal binding site. We now describe structural features of the metal binding site that are not observable by standard NMR methods in native Pdx due to paramagnetic line broadening. These results provide the first example of high-resolution NMR-derived structural data concerning the metal binding domain of an Fe2S2 ferredoxin, and the first structural information of any sort for the metal binding site of a ferredoxin from this class, which includes adrenodoxin, placental ferredoxin and terpredoxin. Assignments were obtained by applying multidimensional NMR methods to a series of selectively and nonselectively 15N- and 13C/15N-labeled GaPdx samples. For most experiments, a mutant of Pdx was used in which a nonligating Cys85 is replaced by serine. All of the major structural features that were identified in native Pdx are conserved in GaPdx. The overall protein dynamics is considerably faster in GaPdx than in the native protein, as reflected by amide proton exchange rates. The C-terminal residue, Trp106, also exhibits considerable mobility, as indicated by 15N[1H] NOE and 15N T1 values of the C-terminal residue of the protein.
Kazanis
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Bioorganic Chemistry Program, Brandeis University, Waltham, MA 02254-9110, USA.
Pochapsky
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D005288:Ferredoxins / Q000737:chemistry* / Q000145:classification; D005708:Gallium / Q000737:chemistry*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008280:Magnetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary
C081714:2Fe-2S ferredoxin; D002247:Carbon Isotopes; D005288:Ferredoxins; D009587:Nitrogen Isotopes; C100137:putidaredoxin; D006859:Hydrogen; D005708:Gallium