Electron paramagnetic resonance and optical evidence for interaction between siroheme and the tetranuclear iron-sulfur center (Fe4S4) prosthetic groups in complexes of Escherichia coli sulfite reductase hemoprotein with added ligands

Abstract

The EPR spectrum of the reduced Fe4S4 center (S = 1/2) in fully reduced native (unligated) E. coli NADPH-sulfite reductase hemoprotein subunit (SiR-HP) is perturbed by interaction with paramagnetic ferrous siroheme (S = 1 or 2) to yield several novel sets of EPR signals: one set with all g values between 2.0 and 2.8, termed the S = 1/2 type, and 2 sets with the lowest field g value between 4.7 and 5.4, termed the S = 3/2 type. The EPR spectra of fully reduced SiR-HP are nearly quantitatively converted to the classical g = 1.94 type typical of S = 1/2 Fe4S4 clusters when the heme has been ligated by strong field ligands such as CO, CN-, S2- and AsO2-, converting the ferroheme to S. = 0. The exact line shapes and g values of the g = 1.94 differ markedly when different ligands are bound to the heme. Also, optical difference spectra taken between enzyme species in which the heme is kept in the same (Fe2+) oxidation state while the Fe4S4 center is reduced or oxidized show that the optical spectrum of the ligated siroheme is sensitive to the oxidation state of the Fe4S4 cluster. The heme-Fe4S4 interaction of native SiR-HP apparently persists even when the heme Fe is bound to exogenous ligands. The g values of the exchange-coupled S = 1/2 and S = 3/2 type signals of native reduced SiR-HP can be significantly shifted by addition of potential weak field heme ligands.sbd.halides and formate.sbd.or low concentrations of certain chaotropic agents.sbd.guanidinium salts and dimethyl sulfoxide.sbd.to the fully reduced enzyme. Such agents can also promote interconversion of the S = 1/2 and S = 3/2 type signals. These effects are reversed on removal of the agent. Treatment of reduced SiR-HP with relatively large concentrations of chaotropes, e.g., 60% dimethyl sulfoxide or 2 or 3 M urea, leads to abolition of the S. = 1/2 and S = 3/2 EPR signals and their replacement by signals of the g = 1.94 type.