Formation and characterization of an all-ferrous Rieske cluster and stabilization of the [2Fe-2S]0 core by protonation

The all-ferrous Rieske cluster, [2Fe-2S]⁰, has been produced in solution and characterized by protein-film voltammetry and UV–visible, EPR, and Mössbauer spectroscopies. The [2Fe-2S]⁰ cluster, in the overexpressed soluble domain of the Rieske protein from the bovine cytochrome bc₁ complex, is formed at –0.73 V at pH 7. Therefore, at pH 7, the [2Fe-2S]^1+/0 couple is 1.0 V below the [2Fe-2S]^2+/1+ couple. The two cluster-bound ferrous irons are both high spin (S = 2), and they are coupled antiferromagnetically (–J ≥ 30 cm^–1, H =–2JS1·S2) to give a diamagnetic (S = 0) ground state. The ability of the Rieske cluster to exist in three oxidation states (2+, 1+, and 0) without an accompanying coupled reaction, such as a conformational change or protonation, is highly unusual. However, uncoupled reduction to the [2Fe-2S]⁰ state occurs at pH > 9.8 only, and at high pH the intact cluster persists in solution for <1 min. At pH < 9.8, the all-ferrous cluster is stabilized significantly by protonation. A combination of experimental data and calculations based on density functional theory suggests strongly that the proton binds to one of the cluster μ₂-sulfides, consistent with observations that reduced [3Fe-4S] clusters are protonated also. The implications for our understanding of coupled reactions at iron–sulfur clusters and of the factors that determine the relative stabilities of their different oxidation states are discussed.Iron–sulfur (FeS) clusters are essential to all forms of life. Most frequently, they are simple electron carriers, but they also constitute catalytic centers, structural scaffolds, and sensors, and they undergo oxidative degradation (1, 2). The rhombic [2Fe-2S], cuboidal [3Fe-4S], and cubane [4Fe-4S] clusters are the most common, but elaboration of these basic modules has produced clusters that contain heterometals and up to eight iron centers. For example, the catalytic clusters in acetyl-CoA synthase contain nickel, and the P-cluster and iron–molybdenum cofactor of nitrogenase can be considered to be two cuboidal subclusters joined by sulfurs. Assembly, disassembly, and interconversion of the simpler clusters are exploited in oxygen sensing and in the control of intracellular iron levels, and they also constitute enzyme active sites, such as in aconitase, chloroplast ferredoxin:thioredoxin reductase, and biotin synthase. In contrast, uncontrolled cluster disassembly (during oxidative stress) accelerates the production of reactive oxygen species and therefore is potentially very damaging.Formally, each iron center in a cluster can be ferric or ferrous, so [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters have three, four, and five possible oxidation states, respectively. In proteins, the oxidation states of [3Fe-4S] clusters cover the widest range because many have been observed in the 1+ (all-ferric), 0, and 2–(all-ferrous) states (3). Formation of the [3Fe-4S]⁰ state is associated with protonation (4), and reduction to the 2–state occurs only upon the uptake of a total of three protons (3). Therefore, the overall charge is conserved, and it is likely that [3Fe-4S] clusters exist in more than two oxidation states because they can be protonated. The protons probably bind on the three μ₂-sulfides, consistent with the ability of [3Fe-4S]⁰ clusters to coordinate a fourth metal ion (5). In contrast, most [4Fe-4S] clusters in proteins are confined to only two oxidation states, either the 2+ and 1+ states or the 3+ and 2+ states in high-potential iron proteins. The [4Fe-4S] cluster in the Fe-protein of nitrogenase (“the Fe-protein”) is unique because it can exist in the all-ferrous state and is stable in more than two oxidation states (2+,1+, and 0) (6). This versatility may be because of its unusually high solvent accessibility (7), although during turnover it undergoes significant conformational changes coupled to nucleotide binding (8), and protonation of the all-ferrous state has not been excluded. The nitrogenase [8Fe-7S] P-cluster (“the P cluster”), the only high-nuclearity cluster not involved directly in small molecule activation, also exists in three oxidation states, but interconversions between them are coupled to changes in cluster structure and ligation and to protonation; the most reduced state may be all-ferrous (9, 10). There is no confirmed example of an FeS cluster in a protein undergoing sequential uncoupled redox transformations.Under physiological conditions, [2Fe-2S] clusters have been observed in the 2+ and 1+ states only. The all-ferrous state may not have been observed because the potential for its formation is too negative, the all-ferrous cluster is unstable, or two-electron reduction is precluded in the absence of a charge-compensation mechanism. In contrast, in anaerobic nonaqueous solvents, synthetic [2Fe-2S] analogues can be reduced sequentially by two electrons (11); the reduction potentials are reported to be only ≈0.25 V apart, but the proposed all-ferrous nature of the fully reduced state has not been demonstrated. A [2Fe-2S]⁰ cluster was generated artificially in spinach ferredoxin by irreversible complexation of the protein to a chromium reductant, increasing the reduction potential significantly and adding extra positive charge (12). Voltammetric signals from the soluble Rieske domain from the bovine heart cytochrome bc₁ complex (–0.84 V at pH 7, potential reported to be pH independent) were attributed to formation of the all-ferrous cluster, but no characterization was attempted (13). Here, we describe the reversible formation of a stable, unmodified [2Fe-2S]⁰ cluster-containing protein and its extensive characterization. The [2Fe-2S]⁰ cluster is a Rieske cluster, so its formation is facilitated by the neutral, electronegative histidine ligands. Our results provide insight into the potentials, stabilities, and reactivities of the different oxidation states of the simplest FeS clusters and are also relevant to understanding the structures and functions of higher nuclearity clusters.