Spectroscopic characterization of a high‐potential monohaem cytochrome from Wolinella succinogenes , a nitrate‐respiring organism

When purified, a high‐potential c ‐type monohaem cytochrome from the nitrate‐respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between‐two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem‐methionine coordination (low‐spin form) coexisting with a high‐spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid‐point redox potential measured by visible redox titration of the low‐spin form was approximately +100 mV. Binding cyanide ( K a = 5 × 10 5 M −1 ) resulted in the displacement of the methionyl axial residue, and full conversion to a low‐spin, cyanide‐bound form. Structural features were studied by 300‐MHz 1 H‐NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5–10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high‐spin/low‐spin equilibrium with a transition p K a of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25°C) was extremely temperature‐dependent [δ(323K) –δ(273K) = 7.2 ppm] and was assigned to the S‐CH 3 group of the axial methionine. In the ferrous state only a low‐spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR‐redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 × 10 6 M −1 s −1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.