ABSTRACT
Many of the iron-sulfur proteins described in the previous paragraphs work as elec tron transfer proteins. Several of them shuttle electrons from photosynthetic systems to soluble or membrane-bound acceptors in archaea, eubacteria, and eukaryotes. However, the electron transfer role of these proteins is not necessarily related to photosynthesis (see, for instance, the ferredoxins from Clostridium pasteurianum). Besides minor structural differences due to evolution, proteins belonging to different structural superfamilies have been chosen by nature to fulfill a common electron transfer function, by exploiting the ability of the protein moiety (1) to recognize the physiological redox partner, (2) to provide the intramolecular pathway from the pro tein surface to the metal cluster at the appropriate transfer rate, and (3) to tune the reduction potential of the redox center to the value required for a particular reduction step in a complex electron pathway. It is worth noting here that iron-sulfur proteins host different types of metal clusters, but the metal center is not necessarily the origin of variability of the reduction potential among different proteins. In fact, the intrinsic reduction potential of a particular iron-sulfur cluster is always modulated by the protein moiety as illustrated in Fig. 25: the reduction potentials cover a range of hundreds of millivolts within classes of proteins with the same type of cluster. Moreover, the effect of the polypeptide chain on the reduction potential of the cluster appears remarkable in comparison of its values in synthetic analogues with those found in corresponding proteins. For example, the [Fe4S4]3+/2+ couple shows a range of +500 to +90 mV in HiPIPs vs. —150 mV in model compounds, and the [Fe4S4]2+/+ couple shows a range of —650 to —280 mV in ferredoxins vs. —1200 mV in model compounds [10]. More importantly, the protein moiety is also responsible for the accessible oxidation states of polymetallic clusters in proteins dissolved in aqueous solutions, even though the oxidation state of each iron ion can only vary between +3 and +2, and the cluster should, in principle, be able to accept one electron per iron ion. Both +3 and +2 states are paramagnetic high spin, with S = 5/2 and S = 2, respectively. Therefore, magnetic coupling within the clusters always occurs (for a comprehensive review, see [124]). The coupling is always antiferromagnetic, and therefore the total S value of the cluster is always the lowest possible. Table 6 sum marizes the situation for the most common clusters. For clusters with more than two iron ions, partial coupling of two individual spins leads to the occurrence of subspins, which may be high spin and display the phenomenon of valence delocalization. For example, the [Fe4S4]3+ cluster can be described as a pair of ferric ions (integer sub spin) coupled to a pair of mixed-valence Fe2 5+ ions (semi-integer subspin). Within a certain cluster, chemical equilibria may exist among species differing in the delocali zation of the mixed-valence pair [10]. The protein control over the reduction potential of the cluster extends also to control of the microscopic reduction potentials of indi vidual iron ions or subspin pairs. For instance, in plant-type 2Fe-2S ferredoxins, the
TABLE 6
Spin Distribution within Iron-Sulfur Clusters
Cluster Individual Spins Subspins3 Total spin
[Fe2S2]2+ 5/2, 5/2 5/2, 5/2 0 [Fe2S2]+ 2, 5/2 2, 5/2 1/2 [Fe3S4]° 2, 5/2, 5/2 9/2*, 5/2 2 [Fe3S4]+ 5/2, 5/2, 5/2 2, 5/2 (3, 5/2) 1/2 [Fe4S4]° 2, 2, 2, 2 0, 0 (?) 0 [Fe4S4]+ 2, 2, 2, 5/2 9/2*, 4 (?) 1/2 [Fe4S4]2+ 2, 2, 5/2, 5/2 9/2*, 9/2* (?) 0 [Fe4S4]3+ 2, 5/2, 5/2, 5/2 9/2*, 4 (7/2*, 3)
(9/2*, 4 + 7/2*, 4) 1/2
couple [Fe2S2]2+/+ is the only one that is accessible in water, i.e., only one of the two iron ions can be reduced. In these systems, localization of the ferrous ion within the peptide frame is strictly limited to the iron closer to the protein surface, as demon strated by NMR [125]. Thus, the individual reduction potentials of each iron ion are no longer equivalent because the cluster symmetry is broken by the protein environ ment. Antiferromagnetic coupling between the two iron ions prevents electron delo calization in the reduced Fe2S2 cluster. An [Fe2S2]° cluster has been recently obtained by using a chromium(II) complex as a reductant. The complex binds the protein and remains bound after reduction as an inert chromium(III) adduct, whose positive charge contributes to stabilize the superreduced [Fe2S2]° cluster [126,127]. The most convincing explanation of the role of the protein in this control takes into account the different dielectric constants characterizing the protein medium and the bulk solvent water. Starting from the simplest iron-sulfur proteins, rubredoxins have much more positive reduction potentials in water (-60 to -1-43 mV; dielectric constant of 80.2) than their synthetic analogue Fe(S2-o-xylyl)2]_ in DMF (-1030 mV; dielectric constant of 36.7). Since the addition of one more electron to the RS4 metal center further increases its negative charge, only a highly polar solvent can stabilize the reduced state, resulting in a lower energy variation during this endoenthalpic process. In this case, the surrounding protein moiety does not completely shield the metal center from access by water molecules, thus maintaining a considerable solvent effect. In HiPIPs, the [Fe4S4] cluster is well embedded in the polypeptide chain, whereas the same type of cluster in bacterial ferredoxins is more exposed to solvent. This feature contributes to the large difference in reduction potential between these two classes of proteins, which only use the [Fe4S4]3+/2+ or the [Fe4S4]2+/+ couple, respectively. Only a fast and reversible conformational change makes the superreduced [Fe4S4]+ state accessible at very low reduction potential (-0.64 V) in the Rhodopila globiformis HiPIP, a protein with one of the highest reduction potentials associated to the couple [Fe4S4]3+/2+ (-1-0.45 V) (Figs. 25 and 26, below). Besides solvent effects modulated by the protein, there are other direct mechanisms with which the polypeptide chain can control the reduction potential of iron-sulfur clusters and generally of metal centers. The availability of a large number of high-resolution three-dimensional structures, both in the solid state and in solution, and the possibility of introducing point muta tions have provided powerful tools to discern single contributions to the observed reduction potential. Of course, a direct comparison between protein structures and redox properties is reasonable only within each class of iron-sulfur proteins, since numerous effects contribute simultaneously to the large variations between classes. Starting from the metal center and moving toward the protein surface, the cluster senses the influence (1) of the protein ligands through differences in the coordination geometry, (2) of the backbone NH dipoles surrounding it, some of them pointing directly to sulfur atoms of either sulfide or cysteine ligands, and (3) of the unit charges provided by ionizable amino acids located at the protein surface, whose interaction with the cluster is to a large extent attenuated by the solvent shielding effect. This influence is in all cases electrostatic. Ligand effects mainly lead to different distribu tions of the electron density among the cluster ions through different degrees of
overlap between the orbitals of the donor atom and those of the coordinated iron ions. These effects are probably not dramatic, as even a change in a donor atom from sulfur to oxygen in a Cys-to-Ser point mutant of C. vinosum HiPIP has led to a lowering of the reduction potential of 25 mV only [128]. The shift in potential is independent from the sequence position of the mutated ligand and thus also from the position of the iron ion in the protein frame. Indeed, both C77S and C64S variants of C. vinosum HiPIP (i.e., mutations of the third and fourth cysteine ligand to the cluster) experience an effect of the same extent on the reduction potential [129]. This equivalence in C. vinosum HiPIP is mainly due to the comparable individual reduction potentials for each iron ion. They were calculated assuming the existence of at least two different electron isomers in fast chemical equilibrium that have been postulated on the basis of the temperature dependence of the hyperfine shifted 1H-NMR resonances of the cysteine ligands [10,130,131]. In proteins in which the iron valence is localized, the picture is different. In the Anabaena 2Fe-2S ferredoxin, the effect of cysteine-to-serine mutations is different for each iron ion [132]. Whereas the reduction potential of C46S (—381 mV) is not significantly different from that of wild type ferredoxin (—384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demon strating that the cluster potential is sensitive to mutations made at the ferric iron in reduced 2Fe-2S Fds with localized valences. For both C. vinosum HiPIP and Anabaena ferredoxin, X-ray or NMR structures of mutants and wild type proteins have excluded any large molecular reorganization due to the point mutations. In other cases, even smaller changes resulted in disassembly of the cluster as a conse quence of increased solvent accessibility to the cluster and its subsequent hydrolysis (see below). This problem occurs especially when the mutation is aimed at eliminating a backbone amide group that can stabilize, if oriented properly to the metal center, the extra electron added during reduction. Some of these NH dipoles are pointing directly toward sulfur atoms of either cluster sulfide or cysteine ligands. HiPIPs possess five such amide groups and ferredoxins eight. This difference could account for the 600 mV lower reduction potential presented by HiPIP considering the same [Fe4S4]2+/+ reduction step. Only recently, it has been possible to measure in C. vino sum HiPIP, the contribution of a single main-chain amide group that directly points to a sulfur donor atom of a cysteine ligand [133]. The substitution of Ser-79 with proline eliminates one NH backbone amide group without any structural changes in the HiPIP, but this mutant has a reduction potential 100 mV lower than that of wild type protein. Theoretical calculations based on an electrostatic model simulated such a decrease by taking into account only the effect of the disappearance of the dipolecharge interaction, while NMR data excluded any large reorganization of the electron distribution among the cluster ions due to the loss of a hydrogen bond. A similar lowering of the reduction potential has been observed in a Rieske iron-sulfur protein as a consequence of a point mutation eliminating an OH dipole located at the side chain of a polar residue [134]. Removal, by site-directed mutagenesis, of the hydroxyl group of Ser-183 pointing to the sulfide ion S-l of the cluster in the Saccharomyces cerevisiae Rieske iron-sulfur protein lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydroxyl group of Tyr-185 close to the Sy of Cys-159
lowers the midpoint potential by 65 mV. Elimination of both hydroxyl groups has an approximately additive effect, lowering the midpoint potential by 180 mV. Solvent exposure is low and similar for both OH dipoles, and the differences in the observed effects have not been discussed by the authors [134]. A tentative explanation could be found in the different distances between the dipole vectors and the reducible iron ion, with the latter being the histidine-coordinated one. For the same reason, surface unit charges associated with ionizable groups of amino acid side chains have a lower interaction energy with the cluster charge than inner dipoles. Moreover, the contri bution of each charge is not additive in plant-type ferredoxins, where the cluster is asymmetrically located inside the protein frame, whereas in HiPIPs, where the clus ter is well embedded in the core of the protein moiety, the effects of each surface unit charge add up to a total contribution that is roughly proportional to the net total charge (Fig. 26). A positive surface net charge will, of course, stabilize the extra electron added upon reduction.
