Resonance Raman Spectroscopic Evidence for the FeS4 and Fe-O-Fe Sites in 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. The RR spectra of rubrerythrin excited at 496.5 and 568.2 nm are dominated by bands similar to those of rubredoxin and conform to the vibrational 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 Desulfovibrio gigas rubredoxin FeS4 site. Similar differences in frequencies of bands 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 cysteinyl ligands in the rubrerythrin versus rubredoxin FeS4 sites. The RR spectrum of rubrerythrin excited at 406.7 nm shows dramatically diminished intensities of the FeS4 bands with concomitant enhancement of a band 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 stretch of a bent oxo-bridged diiron(III) cluster and indicate that this cluster has at least one additional bridging ligand. A small but reproducible 2-cm-1 shift to higher frequency of this symmetric Fe-O-Fe stretch in D2O may indicate involvement of the oxo bridge in hydrogen bonding.