A pure S = 3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin.

The properties of the [4Fe-4S]2+/+ cluster in wild-type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose alpha-C is less than 4 A from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38-mV decrease in the midpoint potential of the [4Fe-4S]2+/+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe-4S]+ cluster in the wild-type protein, 80% S = 3/2 (E/D = 0.22, D = +3.3 cm(-1)) and 20% S = 1/2 (g = 2.10, 1.87, 1.80), is converted into a homogeneous S = 3/2 (E/D = 0.30, D = -0.7 cm(-1)) form in the A33Y variant. As the first example of a pure S = 3/2 [4Fe-4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe-4S]+ clusters.

Effects of mutations in aspartate 14 on the spectroscopic properties of the [Fe3S4]+,0 clusters in Pyrococcus furiosus ferredoxin.

The properties of [Fe(3)S(4)](+,0) clusters in wild-type and mutant forms of Pf Fd with Asp, Ser, Cys, Val, His, Asn, and Tyr residues occupying position 14, i.e., proximal to the three micro(2)-S atoms of the cluster, have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and resonance Raman (RR) spectroscopies. Two distinct types of [Fe(3)S(4)] clusters are identified on the basis of the breadth of the S = (1)/(2) [Fe(3)S(4)](+) EPR resonances and the marked differences in the VTMCD spectra of the S = 2 [Fe(3)S(4)](0) clusters. On the basis of the available NMR data for [Fe(3)S(4)](+, 0) clusters in ferredoxins, the distinctive properties of these two types of [Fe(3)S(4)] clusters are interpreted in terms of different locations of the more strongly coupled pair of irons in the oxidized clusters and the valence-delocalized pair in the reduced clusters. Near-IR VTMCD measurements indicate the presence of S = (9)/(2) valence-delocalized pairs in both types of [Fe(3)S(4)](0) clusters, and the spin-dependent delocalization energies associated with the Fe-Fe interactions were determined to be approximately 4300 cm(-)(1) in both cases. We conclude that the nature of the residue at position 14 in Pyrococcus furiosus ferredoxin is an important determinant of the location of the reducible pair of irons in a [Fe(3)S(4)](+,0) cluster, and the redox properties of the wild-type and mutant ferredoxins are discussed in light of these new results.

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.