A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas.
Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using alpha-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined. High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca. 80% of the protein). A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component.
Mo
H
H
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Pochapsky
S S
SS
Pochapsky
T C
TC
D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D005296:Ferrous Compounds / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011522:Protons; D011549:Pseudomonas / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013455:Sulfur / Q000737:chemistry
D005288:Ferredoxins; D005296:Ferrous Compounds; D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins; D012996:Solutions; C118750:terpredoxin; D013455:Sulfur