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
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- 作者：Phillip S. Brereton ; Randall E. Duderstadt ; Christopher R. Staples ; Michael K. Johnson ; and Michael W. W. Adams
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 10, 1999
- 年：1999
- 卷：38
- 期：32
- 页码：10594 - 10605
- 全文大小：147K
- 年卷期：v.38,no.32(August 10, 1999)
- ISSN：1520-4995
文摘
Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe4S4] cluster coordinated by threecysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14). In this study, thespectroscopic, 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 coordinationsphere of one serine and three cysteine residues, with 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 [Fe4S4]2+,+cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein andEPR 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 changingfrom complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligandin each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited stateproperties for the paramagnetic [Fe4S4]+ centers in each of these variant proteins, with the D14C andD14C/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 
C)of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (Em= -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were morenegative 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 Cys11and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibiteda pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of theserinate ligand to the reduced cluster (pKa = 4.75). All five variants had similar Km and Vm values in acoupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized byferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation ateach Fe atom in the [Fe4S4] cluster appears to have little effect on the interaction and the electron transferbetween Fd and FNOR.
C)of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (Em= -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were morenegative 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 Cys11and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibiteda pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of theserinate ligand to the reduced cluster (pKa = 4.75). All five variants had similar Km and Vm values in acoupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized byferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation ateach Fe atom in the [Fe4S4] cluster appears to have little effect on the interaction and the electron transferbetween Fd and FNOR.