7.4.1: Iron-sulfur Proteins and Models

Iron sulfide proteins involved in electron transfer are called ferredoxins and rubredoxins.* The ferredoxins were discovered first, and were originally classified as bacterial (containing Fe₄S₄ clusters) and plant (containing Fe₂S₂ clusters) ferredoxins. This classification is now recognized as being not generally useful, since both Fe₂S₂ and Fe₄S₄ ferredoxins are found in plants,^14,15 animals,^2,6,16 and bacteria.⁴ Ferredoxins are distinguished from rubredoxins by their possession of acid-labile sulfide; i.e., an inorganic S^2- ion that forms H₂S gas upon denaturation at low pH. Rubredoxins have no acid-labile sulfide, and generally have a single iron in a more or less isolated site. Despite their lack of acid-labile sulfide, rubredoxins are included in this chapter because they have sequences much like those of the ferredoxins, and because their simple mononuclear Fe²⁺ and Fe³⁺ sites provide convenient illustrations of key structural and spectroscopic features.

In most ferredoxins, and in all rubredoxins, the protein ligands are cysteines, which provide four thiolate donors to the 1Fe, 2Fe, or 4Fe center. Additionally, the existence of 3Fe centers and of Fe-S sites that contain a second metal (i.e., heteronuclear clusters) make the Fe-S class a broad and multifunctional one.

Simple cytochromes and simple iron-sulfide proteins are similar, in that both can undergo one-electron transfer processes that are generally uncoupled from proton-, atom-, or group-transfer processes. Some of these proteins, such as cytochrome c₃ from Desulfovibrio with four hemes¹⁷ or ferredoxin from Clostridium pasteurianum with two Fe₄S₄ centers,⁶ can transfer more than one electron, because they have multiple copies of a one-electron transfer group. The cytochromes were discovered in 1886 by McMunn,¹⁸ and their role in metabolism was discovered in the 1920s by Keilin (Chapter 6). The intense optical absorbance of these heme-containing proteins contributed singularly to their discovery and biochemical characterization. In contrast, the iron-sulfur proteins, although red to red-brown, absorb far more weakly in the visible region than do the cytochromes. Their presence is sometimes obscured by the cytochromes, and their frequent air instability made their initial recognition and isolation more difficult. It was not until the early 1960s that discoveries by several research groups¹⁹ led to the isolation, recognition, and characterization of the ferredoxins. The use of EPR spectroscopy and its application to biological systems had a profoundly stimulating effect on the field (see below).

Although cytochromes were discovered first, the ferredoxins are likely to be the older proteins from an evolutionary perspective.²⁰ Ferredoxins have relatively low-molecular-weight polypeptide chains, require no organic prosthetic group, and often lack the more complex amino acids. In fact, the amino-acid composition in clostridial ferredoxin is close to that found in certain meteorites.²¹

The various Fe-S sites found in electron-transfer proteins (ferredoxins) are also found in many enzymes,^6,11,22.23 where these centers are involved in intraor interprotein electron transfer. For example, sulfite reductase contains a siroheme and an Fe₄S₄ center,²⁴ which are strongly coupled and involved in the sixelectron reduction of SO₃^2- to H₂S. Xanthine oxidase (see Figure 7.1) has two identical subunits, each containing two different Fe₂S₂ sites plus molybdenum and FAD sites. In xanthine oxidase, the Mo(VI) site carries out the two-electron oxidation of xanthine to uric acid, being reduced to Mo(IV) in the process.²⁵ The Mo(VI) site is regenerated by transferring electrons, one at a time, to the Fe₂S₂ and flavin sites, thereby readying the Mo site for the next equivalent of xanthine. Although the Fe₂S₂ sites do not directly participate in substrate reactions, they are essential to the overall functioning of the enzyme system. The Fe₂S₂ centers in xanthine oxidase play the same simple electron-transfer role as the Fe₂S₂ ferredoxins play in photosynthesis.

Figure 7.1 - A schematic drawing of xanthine oxidase illustrating the Mo, flavin, and Fe₂S₂ sites and interaction of the enzyme with substrate and oxidant(s).

Structurally, all the iron-sulfur sites characterized to date are built up of (approximately) tetrahedral iron units (see Figure 7.2). In rubredoxins the single iron atom is bound in tetrahedral coordination by four thiolate ligands provided by cysteine side chains. In two-iron ferredoxins the Fe₂S₂ site consists of two tetrahedra doubly bridged through a pair of sulfide ions, i.e., Fe₂(\(\mu_{2}\)-S)₂, with the tetrahedral coordination of each Fe completed by two cysteine thiolates. In four-iron or eight-iron ferredoxins, the 'thiocubane' Fe₄S₄ cluster consists of four tetrahedra sharing edges with triply bridging S^2- ions, i.e., Fe₄(\(\mu_{3}\)-S)₄, with each Fe completing its tetrahedron by binding to a single cysteine thiolate. Finally, for Fe₃S₄ clusters, which are now being found in more and more proteins, the well-established structure has one triply bridging and three doubly bridging sulfide ions, Fe₃(\(\mu_{3}\)-S)(\(\mu_{2}\)S)₃. The Fe₃S₄ unit can be thought of as derived from the 'thiocubane' Fe₄S₄ unit by the removal of a single iron atom.

In what follows we will introduce these structures in the order 1Fe, 2Fe, 4Fe, and 3Fe. For each, we will first discuss the physiological role(s) of the particular proteins, then the structural features, followed by the spectroscopic properties and model systems.

Figure 7.2 - Structural systematics of Fe-S units found in proteins: (A) rubredoxin single Fe center; (B) Fe₂S₂ unit; (C) Fe₄S₄ unit; (d) Fe₃S₄ unit.

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* For review articles, see References 1-11. For a discussion of nomenclature, see References 12 and 13.