title: Redox-dependent 1H NMR spectral features and tertiary structural constraints on the C-terminal region of putidaredoxin.
abstract: Putidaredoxin (Pdx) is a 106-residue Fe2S2 ferredoxin which acts as the physiological reductant and effector of cytochrome P-450cam. Pdx has two accessible oxidation states, Fe+3-Fe+3 (oxidized) and Fe+3-Fe+2 (reduced), and exhibits redox-dependent binding affinities for cytochrome P-450cam, with reduced Pdx binding over 100-fold more tightly than oxidized Pdx to the oxidized cytochrome P-450cam [Hintz, M. J., Mock, D. M., Peterson, L. L., Tuttle, K., & Peterson, J. A. (1982) J. Biol. Chem. 257, 14324-14332]. The analysis of two-dimensional 1H NMR experiments has yielded sequential 1H resonance assignments for the diamagnetic regions of the reduced form of Pdx, which are compared to those of oxidized Pdx, described previously [Ye, X. M., Pochapsky, T. C., & Pochapsky, S. S. (1992) Biochemistry 31, 1961-1968]. Increased unpaired electron-spin density on the metal cluster in reduced relative to oxidized Pdx increases the number of 1H resonances which are broadened by the metal cluster, and the pattern of paramagnetic broadening provides information concerning the placement of the metal cluster within the protein. Two-dimensional exchange experiments on half-reduced samples of Pdx indicate that electron self-exchange is slow on the chemical shift time scale, with a second-order rate constant < or = 66 M-1 s-1 at 290 K. Spectral changes unrelated to increases in unpaired electron-spin density are also observed. The largest changes of this type are observed for features structurally contiguous with the C-terminal region Pro 102-Trp 106. The C-terminal residue Trp 106 has been implicated in binding to cytochrome P-450cam.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
authors.forename: T C
authors.initials: TC
authors.lastname: Pochapsky
authors.forename: G
authors.initials: G
authors.lastname: Ratnaswamy
authors.forename: A
authors.initials: A
authors.lastname: Patera
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D004583:Electrons; D005288:Ferredoxins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D014316:Tritium
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D014316:Tritium; C100137:putidaredoxin