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.

Surface ECE mechanism in protein film voltammetry—a theoretical study under conditions of square-wave voltammetry

“…Of these, special attentions attract the surface redox processes in which both compounds of a given redox couple are strongly immobilized on the electrode surface [1][2][3][4][5][6]. Given all the attention to the surface redox reactions, the application of voltammetry for probing the chemistry of redox proteins has recently emerged as an especially simple and powerful method of investigating biologically relevant redox-active compounds [7][8][9][10]. By simple adsorption of the redox protein sample onto the surface of some suitable lipophilic electrode, insights into the processes of electron transfer and protein-protein interactions can be obtained from experiments performed in common voltammetric set-up [7][8][9][10].…”

“…Given all the attention to the surface redox reactions, the application of voltammetry for probing the chemistry of redox proteins has recently emerged as an especially simple and powerful method of investigating biologically relevant redox-active compounds [7][8][9][10]. By simple adsorption of the redox protein sample onto the surface of some suitable lipophilic electrode, insights into the processes of electron transfer and protein-protein interactions can be obtained from experiments performed in common voltammetric set-up [7][8][9][10]. Very often, the electron transfer steps by various organic compounds and proteins, studied in thin-film or protein-film voltammetric scenarios, are coupled by chemical reactions [1,4,7,11,12].…”

Structure of C42D Azotobacter vinelandii FdI

“…Even though we were successful in converting the 4Fe cluster of C42D FdI to a 3Fe cluster by electrochemical pulsing, the product could not be readily converted back to a 4Fe cluster. Thus, unlike the situation with easily converted clusters (70,98), when films of the 6Fe form of C42D FdI were placed in 5 mM solutions of Fe 2ϩ , Zn 2ϩ , and Tl ϩ , no signs of metal uptake were observed.…”

“…2) with a central aspartate shows that in most cases the residue at position 44 is much smaller and/or not hydrophobic, which may facilitate the movement of the aspartate. An exception is PfFd, which does easily convert, albeit at a slower rate than for DaFdIII (70), and has a hydrophobic Ile at the position homologous to the FdI residue number 44. Thus, although the residue 44 may be important in facilitating the cluster conversion, it cannot be the controlling factor.…”

Electron transfer and coupled processes in protein film voltammetry

“…We have been using PFV to solve problems that include the characterization of labile Fe-S clusters [9-131; mechanisms of rapid, electroncoupled proton transfer [ 7 ] ; redox properties of the catalytic states of peroxidases [14,15]; and the complex catalytic electron-transport characteristics of multi-centred enzymes [8,16- [13,22]. This is a simple example of a cluster transformation and has at least some indirect physiological relevance: little is known about how clusters are assembled or degraded in proteins, and changes in structure that are induced by cellular levels of Fe or oxygen are known to control expression at the transcriptional and translational levels [23-251. In PfFd, as well as in other systems [9,13], metals (M) apart from Fe can be reversibly incorporated to give heterometal cubanes of formula (M3Fe-4SI.…”