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.
Subject(s)
Electrophysiology/methods , Ferredoxins/chemistry , Ferredoxins/metabolism , Pyrococcus furiosus/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Electric Conductivity , Hydrogen-Ion Concentration , Iron/chemistry , Kinetics , Magnetics , Metals/metabolism , Metals/pharmacokinetics , Oxidation-Reduction , Spectrophotometry, Ultraviolet , Spectrum Analysis/methods , Sulfur/chemistry , ThermodynamicsABSTRACT
Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.
