Fe2S2 protein resonance Raman spectra revisited: structural variations among adrenodoxin, ferredoxin, and red paramagnetic protein

Abstract

Resonance Raman spectra (RR) are reported for bovine adrenodoxin (Ado) and ferredoxin (Fd) from Porphyra umbilicalis, with 34S substituted at the bridging positions of the Fe2S2 cluster. All of the Fe-S stretching modes are assigned on the basis of the 34S isotope shifts and the previous analysis of analogue spectra. Appreciable frequency variations are seen, which can be explained on the basis of stronger Fe-S bonds, both bridging and terminal, in the proteins. This strengthening is attributable to protein compression effects or to H-bond interactions which reduced the negative charge on the iron-sulfur complex. In addition the Fe frequencies suggest an alteration in the Fe-S-C-C dihedral angles which induce mixing of the terminal Fe-S stretches with the S-C-C bending coordinate. The RR spectrum of the Porphyra umbilicalis Fd is identical with that of Fd isolated from Spirulina platensis, for which the crystal structure has been reported, as well as that of spinach Fd. RR spectra have also been obtained for a red paramagnetic protein (RPP) from Clostridium pasteurianum. Its frequencies suggest weakened Fe-S bridge bonding. RR spectra of reduced protein have been obtained for Ado and RPP, but not for Fd. The reduced protein spectra differ strongly from those of the oxidized proteins, and can be accounted for on the basis of a 30% reduction of the Fe-S force constants on the Fe11 side of the Fe2S2 cluster.

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

“…There are three to four additional bands in the 280 -320 cm Ϫ1 region whose peak positions are too weak to be determined with accuracy (not labeled in the figure). These spectra suggest a lower symmetry around the Rieske-type [2Fe-2S] cluster core than that around the biological [2Fe-2S] 2ϩ clusters with complete cysteinyl ligation that typically exhibit six or seven bands under similar conditions (17,20,29,39,(53)(54)(55)(56). The major differences in the RR spectra of ARF and sulredoxin are found in the 240 -400 cm Ϫ1 region; interestingly the overall RR features of oxidized ARF are more similar to those reported for the low potential Rieske-type cluster in bacterial phthalate dioxygenase (17,20), which is in line with our recent iron K-edge XAS analysis showing the structural similarity in the coordination environment of the Rieske-type clusters in ARF and bacterial anthranilate dioxygenase (27) (Table I).…”

“…Resonance Raman Characterization of the Oxidized Riesketype Cluster in ARF-Low temperature RR spectroscopy is a sensitive probe for the microenvironment of biological [2Fe-2S] clusters (52)(53)(54). Earlier RR studies on the pH dependence of the oxidized forms of archaeal and bacterial high potential Rieske proteins (as models for components of proton-translocating respiratory complexes) (17,20,29) have been considered to be the primarily spectroscopic supporting source for the common view to assign the pK a,ox near ϳ8, which influences the E m and the visible CD spectrum, to one of the terminal histidinyl ligands (1)(2)(3)(4).…”

“…4). In the RR spectra of biological [2Fe-2S] 2ϩ clusters with one serinate ligand introduced by site-directed mutagenesis (55,56) and those of bacterial biotin synthase (68) and IscU iron-sulfur protein (69) that have been interpreted in terms of partial non-cysteinyl ligation, the B 3u t modes with predominantly Fe-S t stretching characters occur at anomalously high frequencies in the range 289 -302 cm Ϫ1 compared with 281-291 cm Ϫ1 for clusters in the plant-and vertebrate-type [2Fe-2S] ferredoxins with complete cysteinyl ligation (53,54). Notably the overall RR characteristics in the 250 -450 cm Ϫ1 region of the oxidized [2Fe-2S] center in H64C are distinctly different from these cases, showing an anomalously low frequency band at 258 cm Ϫ1 ; the equivalent band in Rieske and Rieske-type proteins at 260 -270 cm Ϫ1 (Fig.…”

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

“…The resonance Raman spectrum at 77 K of the oxidized triple variant differs significantly from that of the wild-type protein (20,21) or other biological [2Fe-2S] 2ϩ clusters with complete cysteinyl ligations (37,38) and clearly indicates the Rd-like tetrahedral coordination geometry of the iron site (Fig. 2, h and i).…”

A New Type 2 Copper Cysteinate Azurin

“…As in superoxide dismutase H46R (22), the combination bands in the H117G/N42C azurin are indicative of two cysteinate ligands. Such combination bands are observed in copper-substituted liver alcohol dehydrogenase (36) and other metal cysteinate proteins with multiple cysteinate ligands such as Cu A (24,26), rubredoxin (37), and ferredoxin (38) as well as in the copper-sulfide cluster Z ox in N 2 O reductase (26) (Table II). 34 S substitution, which can be used, in principle, to identify multiple Cu-S stretching modes, was not successful.…”