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 [Fe4S4] cluster coord inated by three cysteine (at positions 11, 17, and 56) and one aspartate Li gand (at position 14). In this study, the spectroscopic, redox, and functio nal consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutatio ns have been investigated. The four serine variants each contain a potentia l cluster coordination sphere of one serine and three cysteine residues, wi th serine ligation at each of the four Fe sites of the [Fe4S4] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe 4S4](2+,+) cluster as shown by UV-visible absorption and resonance Raman st udies of the oxidized protein and EPR and variable-temperature magnetic cir cular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced prot ein. Changes in both the absorption and resonance Raman spectra are consist ent with changing from complete cysteinyl cluster ligation in the D14C vari ant to three cysteines' and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state p roperties for the paramagnetic [Fe4S4](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = 1/2 g round states and the D14S,D14C/C17S,and D14C/C56S variants having mixed-spi n, S = 1/2 and 3/2 ground states. The midpoint potentials (pH 7.0, 23 degre es C) of the D14C/C11S and D14C/C17S variants were unchanged compared to th at of the D14C variant (E-m = -427 mV) within experimental error, but the p otentials 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 Fe2.5+Fe2.5+ pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible vale nce-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependen t redox potential over the range of 3.5-10; and this is attributed to proto nation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All fi ve variants had similar K-m and Vm values in a coupled assay in which Fd wa s reduced by pyruvate ferredoxin oxidoreductase (POR) and,oxidized by ferre doxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, th e mode of Ligation at each Fe atom in the [Fe4S4] cluster appears to have l ittle effect on the interaction and the electron transfer between Fd and FN OR.