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Voltammetric studies of the reactions of iron-sulphur clusters ([3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin.

ABSTRACT: Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] adducts co-ordinated in the ferredoxin from the hyperthermophile Pyrococcus furiosus have been studied by protein-film voltammetry, bulk-solution voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S]+/0 and [3Fe-4S]0/2-. Film voltammetry is possible over a wide pH range (2-8), revealing that the [3Fe-4S]+/0 couple shows a complex pH dependence with pKred1=2.8, pKox=4.9 and pKred2=6.7. From MCD, pKred1 corresponds with protonation of [3Fe-4S]0 to give a spectroscopically distinct species, as reported for ferredoxins from Azotobacter and Sulfolobus. The status of the disulphide/disulphydryl entity makes no significant difference to the data (given for the -S-S- form). Formation of the hyper-reduced [3Fe-4S]2- state is observed, requiring 3H+ for the overall 3e- reduction of [3Fe-4S]+, the change therefore being electroneutral. By comparison with the ferredoxin from Desulfovibrio africanus, uptake of Fe(II) and other M(II) by [3Fe-4S]0 to give [M3Fe-4S] clusters is slow (t1/2>10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t1/2<<1 s), suggesting that co-ordination of Tl does not require reorganization of the protein structure. Rates of formation of [3Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involves a labile 'super-oxidized' [M3Fe-4S]3+ state.

SUBMITTER: Fawcett SE

PROVIDER: S-EPMC1219790 | biostudies-other | 1998 Oct

REPOSITORIES: biostudies-other

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Project description:The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single [4Fe-4S]1+,2+ cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys. The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli. While the recombinant form 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 spectroscopy and electrochemistry. The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (Em -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, Em -203 mV). The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation. The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain. With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate. While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated DeltaS degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14. In contrast, there was a linear correlation between the DeltaH degrees' value and reduction potential. Kinetic analyses were carried out at 80 degrees C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P. furiosus). The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems. However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from POR and donate them to FNOR.

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Voltammetric studies of the reactions of iron-sulphur clusters ([3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin.

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