Characterization of the pH-Dependent Resonance Raman Transitions of Archaeal and Bacterial Rieske [2Fe−2S] Proteins

Abstract

The pH-dependent resonance Raman (RR) spectral changes of the cytochrome bc1-associated, high-potential Rieske proteins have frequently been invoked to explain the redox-linked ionization behavior. We report herein RR spectral data of archaeal and bacterial Rieske proteins that directly demonstrate the pH-dependent changes near and above pKa,ox2, but not around pKa,ox1, of the visible circular dichroism (CD) transitions. The RR spectral changes are attributed to modification of the immediate [2Fe-2S] cluster environment due to deprotonation of some exchangeable amide groups in the polypeptide backbone, rather than previously assumed simple changes of the Fe-Nimid stretching vibrations.

Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy – interpretation by molecular mechanics

“…In a previous normal mode calculation, Urushiyama and coworkers have argued that in rubredoxin [50], as well as ferredoxins [51], the Fe-S modes are 'extensively coupled to deformations of the polypeptide backbone'. Significant coupling of the Fe-S stretch with cysteine backbone deformations was also deduced from 1-2 cm À1 shifts after 15 N-labeling of the cysteine nitrogens [52].…”

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).…”

“…Purification of each recombinant holoprotein having a hexahistidinetag at the N terminus was performed as described previously (20,21), except that the heat treatment step (at 65°C for 15-30 min) was omitted for the H44C, H44I/K45C, and double (H44C/H64C) mutants. The recombinant holoprotein was further purified by Sephadex G-75 gel filtration column chromatography (Amersham Biosciences).…”

“…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. Low temperature resonance Raman spectra were recorded at 77 K using a Spex 750M Raman spectrometer fitted with a Spectrum-One 2048 ϫ 512 CCD camera and a Spectra-Physics 2017 Ar ϩ laser (output, 500 milliwatt) by collecting 45°backscattering off the surface of a frozen sample in a quartz microcell (21). The slit width of the spectrometer is 80 m, and a multiscan signal-averaging technique was employed to improve the signal-to-noise ratio.…”

Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

“…The 250-cm Ϫ1 band would have a major contribution from symmetric stretching of the two Fe-N (His) bonds, with one or both of the 282 and 296 cm Ϫ1 bands having a significant contribution from asymmetric stretching of the two Fe-N(His) bonds. The available pH dependence and N-isotope shift data for the Rieske-type and mitoNEET proteins argues against the assignment of the band in the 250 -300 cm Ϫ1 region to pure Fe-N(His) stretching mode (42)(43)(44)(45). Rather, Fe-N(His) stretching is distributed over low energy Fe-S stretching modes and internal modes of coordinated cysteine ligands and enhanced via the visible S-to-Fe charge transfer transitions.…”