Abstract
Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.
Highlights
Natural selection allows the utilization of a limited number of protein scaffolds to produce proteins with different types of active sites for various biological catalysis, molecular recognition, and metabolic needs
We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds
We have recently addressed the influence of substitution of each of two histidine ligands (His-44 and His-64) by cysteine on the properties of a low potential Rieske-type cluster in archaeal Rieske-type ferredoxin (ARF) from the hyperthermophile Sulfolobus solfataricus strain P-1 (19 –22) as a new tractable model
Summary
Natural selection allows the utilization of a limited number of protein scaffolds to produce proteins with different types of active sites for various biological catalysis, molecular recognition, and metabolic needs. The mononuclear iron core is the simplest form of Fe-S redox sites, present in small modular proteins such as rubredoxin (Rd) and desulforedoxin These proteins have the iron atom coordinated in an approximately tetrahedral geometry to the sulfur atoms of four cysteinyl residues [1]. Other major forms of protein-bound Fe-S redox sites are polynuclear clusters (such as [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters) for which some specific synthesis/assembly enzymes are required for in vivo cluster formation and maturation steps [7,8,9,10] These sites are found within complex metalloprotein molecules themselves where they form part of the internal electron transfer conduit to or from the catalytic site (e.g. in some membrane-bound respiratory complexes [11,12,13]) as a result of modular evolution. The replacement of one of the histidine ligands, His-64, by cysteine allowed the assembly of a new low potential [2Fe-2S] cluster with one histidine plus three cysteine ligands in the archaeal Rieske-type protein scaffold, whereas replacement of the other
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