RESONANCE RAMAN-SPECTROSCOPIC EVIDENCE FOR THE FES4 AND FE-O-FE SITESIN RUBRERYTHRIN FROM DESULFOVIBRIO-VULGARIS

Resonance Raman (RR) spectra of the non-heme iron protein rubrerythrin from Desulfovibrio vulgaris unequivocally demonstrate the presence of both a rubredoxin-type FeS4 site and a (mu-oxo)diiron(III) cluster. T he RR spectra of rubrerythrin excited at 496.5 and 568.2 nm are domina ted by bands similar to those of rubredoxin and conform to the vibrati onal pattern expected for a distorted FeS4 tetrahedron of an Fe(S-Cys) 4 site. Numerous overtone and combination bands of the Fe-S stretches are also observed, and a band at 650 cm-1 is assigned to a cysteine C- S stretching mode. The 374-, 355-, and 340-cm-1 bands, assigned to the three components of the nu3(T2) asymmetric FeS4 stretching mode, are 2-8 cm-1 lower than the corresponding frequencies for the Desulfovibri o gigas rubredoxin FeS4 site. Similar differences in frequencies of ba nds assigned to SFeS bending modes between rubredoxin and rubrerythrin are also detected. These frequency differences imply either slightly weaker Fe-S bonds or subtle conformational differences among the cyste inyl ligands in the rubrerythrin versus rubredoxin FeS4 sites. The RR spectrum of rubrerythrin excited at 406.7 nm shows dramatically dimini shed intensities of the FeS4 bands with concomitant enhancement of a b and at 514 cm-1. This band shifts 18 cm-1 to lower frequency when the protein is dissolved in (H2O)-O-18. The frequency of this band and the O-18 isotope shift are those expected for the symmetric Fe-O-Fe stret ch of a bent oxo-bridged diiron(III) cluster and indicate that this cl uster has at least one additional bridging ligand. A small but reprodu cible 2-cm-1 shift to higher frequency of this symmetric Fe-O-Fe stret ch in D2O may indicate involvement of the oxo bridge in hydrogen bondi ng.