| Abstract
| - The ferredoxin (7.5 kDa) of the hyperthermophilicarchaeon, Pyrococcus furiosus, contains asingle [4Fe-4S]1+,2+ cluster that is coordinated bythree Cys and one Asp residue rather than the expectedfour Cys. The role of this Asp residue was investigated using aseries of mutants, D14X, where X = C,S, H, N, V, and Y, prepared by heterologous gene expression inEscherichia coli. While therecombinantform of the wild-type and the D14S and D14C mutants contained a[4Fe-4S]1+,2+ cluster, the D14V,D14H, D14Y, and D14N proteins contained a [3Fe-4S]0,+center, as determined by visible spectroscopyand electrochemistry. The redox potentials (at pH 7.0, 23 °C) ofthe D14C and D14S mutants weredecreased by 58 and 133 mV, respectively, compared to those of thewild-type 4Fe-ferredoxin (Em −368mV), while those of the 3Fe-protein mutants (including the 3Fe-form ofthe D14S, generated by chemicaloxidation) were between 15 and 118 mV more positive than that ofwild-type 3Fe-form (obtained bychemical oxidation, Em −203 mV). Thereduction potentials of all of the 3Fe-forms, except theD14Smutant, showed a pH response over the range 3.0−10.0 with apK of 3.3−4.7, and this was assigned tocluster protonation. The D14H mutant and the wild-type3Fe-proteins showed an additional pK (both at5.9) assumed to arise from protonation of the amino acid side chain.With the 4Fe-proteins, there was nodramatic change in the potentials of the wild-type or D14C form, whilethe pH response of the D14Smutant (pK 4.75) was ascribed to protonation of theserinate. While the ferredoxin variants exhibited arange of thermal stabilities (measured at 80 °C, pH 2.5), none ofthem showed any temperature-dependenttransitions (0−80 °C) in their reduction potentials, and there wasno correlation between the calculatedΔS°‘ values and the absorbance maximum, reductionpotential, or hydrophobicity of residue 14. Incontrast,there was a linear correlation between the ΔH°‘ valueand reduction potential. Kinetic analyses werecarried out at 80 °C using the ferredoxin as either an electronacceptor to pyruvate oxidoreductase (POR)or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, bothfrom P. furiosus). The datashowed that the reduction potential of the ferredoxin, rather thancluster type or the nature of the residueat position 14, appears to be the predominant factor in determiningefficiency of electron transfer in bothsystems. However, compared to all the variants, the reductionpotential of WT Fd makes it the mostappropriate protein to both accept electrons from POR and donate themto FNOR.
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