Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties.
Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe(4)S(4)] cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14). In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated. The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the [Fe(4)S(4)] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe(4)S(4)](2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein. Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic [Fe(4)S(4)](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = (1)/(2) ground states and the D14S, D14C/C17S, and D14C/C56S variants having mixed-spin, S = (1)/(2) and (3)/(2) ground states. The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E(m) = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively. Since the VTMCD spectra indicate the presence of a valence-delocalized Fe(2. 5+)Fe(2.5+) pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All five variants had similar K(m) and V(m) values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation at each Fe atom in the [Fe(4)S(4)] cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR.
Brereton
P S
PS
Department of Biochemistry, Center for Metalloenzyme Studies, University of Georgia, Athens 30602, USA.
Duderstadt
R E
RE
Staples
C R
CR
Johnson
M K
MK
Adams
M W
MW
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D020027:Pyrococcus furiosus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*
D005288:Ferredoxins; D008024:Ligands; D001224:Aspartic Acid; D012694:Serine; D013455:Sulfur; D007501:Iron; D005287:Ferredoxin-NADP Reductase; D003545:Cysteine