S= 9/2 EPR signals are evidence against coupling between the siroheme and the Fe/S cluster prosthetic groups in Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase

Abstract

Sulfite reductases contain siroheme and iron-sulfur cluster prosthetic groups. The two groups are believed to be structurally linked via a single, common ligand. This chemical model is based on a magnetic model for the oxidized enzyme in which all participating iron ions are exchange coupled. This description leads to two serious discrepancies. Although the iron-sulfur cluster is assumed to be a diamagnetic cubane, [4Fe–4S]²⁺, all iron appears to be paramagnetic in Mössbauer spectroscopy. On the other hand, EPR spectroscopy has failed to detect anything but a single high-spin heme. We have re-addressed this problem by searching for new EPR spectroscopic clues in concentrated samples of dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). We have found several novel signals with effective g values of 17, 15.1, 11.7, 9.4, 9.0, 4. The signals are interpreted in terms of an S= 9/2 system with spin-Hamiltonian parameters g= 2.00, D=−0.56 cm⁻¹, |E/D|= 0.13 for the major component. In a reductive titration with sodium borohydride the spectrum disappears with E_m=−205 mV at pH 7.5. Contrarily, the major high-spin siroheme component has S= 5/2, g= 1.99, D=+9 cm⁻¹, |E/D|= 0.042, and E_m=−295 mV. The sum of all siroheme signals integrates to 0.2 spin/half molecule, indicating considerable demetallation of this prosthetic group. Rigorous quantification procedures for S= 9/2 are not available, however, estimation by an approximate method indicates 0.6 S= 9/2 spin/half molecule. The S= 9/2 system is ascribed to an iron-sulfur cluster. It follows that this cluster is probably not a cubane, is not necessarily exchange-coupled to the siroheme, and, therefore, is not necessarily structurally close to the siroheme. It is suggested that this iron-sulfur prosthetic group has a novel structure suitable for functioning in multiple electron transfer.