Spectroscopic Investigation of Selective Cluster Conversion of Archaeal Zinc-containing Ferredoxin fromSulfolobus sp. Strain 7

Abstract

Ferredoxins, small iron-sulfur (FeS) proteins in archaea, serve as water-soluble electron acceptors of acyl-coenzyme A forming 2-oxoacid:ferredoxin oxidoreductase, a key enzyme involved in the central archaeal metabolic pathways (1-5). The 2.0-Å resolution x-ray crystal structure (PDB 1 entry 1XER ; Ref. 6) of ferredoxin from Sulfolobus sp. strain 7 (JCM 10545; optimal growth conditions, pH 2.5-3 and 80°C) showed the presence of an unexpected isolated zinc center, tetrahedrally coordinated by three nitrogen atoms from histidine residues in the N-terminal extension region (N␦1 of His 16 , N⑀2 of His 19 , and N␦1 of His 34 ) and one O␦1 atom from Asp 76 in the FeS cluster-binding core fold (Fig. 1). A similar zinc site was also found in ferredoxin from Thermoplasma acidophilum strain HO-62 that also contained one 1ϩ,0 cluster, and one [4Fe-4S] 2ϩ,1ϩ cluster (7). This site was analyzed by zinc K-edge x-ray absorption spectroscopy (XAS) (8) and was found to be essentially identical to the zinc site in Sulfolobus sp. ferredoxin. Thus, these unusual ferredoxins contain both the conventional FeS clusters and a structurally conserved, isolated zinc center. This new class of bacterial type ferredoxin, isolated from phylogenetically diverse members of several aerobic and thermoacidophilic archaea, are thus called "zinc-containing ferredoxins" (2, 6 -10).Another unexpected result of the crystal structure of airoxidized Sulfolobus sp. zinc-containing ferredoxin was the presence of two [3Fe-4S] clusters beside one isolated zinc center (9) (Fig. 1). The number and type of FeS clusters in this structure are inconsistent with our previous spectroscopic analysis of the purified protein, which suggested one 1ϩ,0 cluster (cluster I; E 1 ⁄2 ϭ Ϫ280 mV) and one 2ϩ,1ϩ cluster (cluster II; E 1 ⁄2 ϭ Ϫ530 mV) (5). Other archaeal zinc-containing ferredoxins from T. acidophilum (7,8) (Fig. 1).Because FeS clusters have a remarkable facility for interconversion under protein-bound conditions (reviewed in Ref. 14), the significant discrepancy of the types of FeS clusters of Sulfolobus sp. zinc-containing ferredoxin in the crystalline and as-isolated states may represent selective degradation of the cluster II to a [3Fe-4S] form in vitro. Although the [4Fe-4S] 7 [3Fe-4S] cluster interconversion is well known in some bacterial type ferredoxins (14 -25) and aconitase (26 -29), there is no conclusive report demonstrating the selective cluster conversion at the cluster II site in bacterial type dicluster ferredoxins. The most extensive spectroscopic analyses of oxidative degra-

Engineering a Three-cysteine, One-histidine Ligand Environment into a New Hyperthermophilic Archaeal Rieske-type [2Fe-2S] Ferredoxin from Sulfolobus solfataricus

“…Energies were calibrated using an internal iron foil standard, assigning the first inflection point to 7111.3 eV. All other data collection parameters were as reported previously (40,41).…”

“…4 Calibration and background removal were performed according to established procedures (40,42). Theoretical amplitude and phase shift functions were calculated using the ab initio code FEFF 8.2 (43).…”

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

“…Energies were calibrated using an internal iron foil standard, assigning the first inflection point to 7111.3 eV. All other data collection parameters were as reported previously (25,26).…”

“…EPR measurements were performed by using a JEX-RE3X spectrometer equipped with an ES-CT470 Heli-Tran cryostat system and a Scientific Instruments digital temperature indicator/controller Model 9650 (25). Spin concentrations of purified recombinant proteins were estimated by double integration, with Cu-EDTA (0.1 mM) and Sulfolobus tokodaii sulredoxin (0.05 mM) (28) as standards.…”

“…Calibration and background removal were performed according to established procedures (25,27). The theoretical amplitude and phase shift functions were calculated using the ab initio code FEFF 8.2 (28).…”