NMR and electron‐paramagnetic‐resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase)

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Abstract
A diheme cytochrome (MW = 37,400) with cytochrome c peroxidase activity was purified from P. stuzeri (ATCC 11607). The heme redox potentials are far apart; 1 of the hemes is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound hemes were probed by visible, NMR and EPR spectroscopies in 3 oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of 3 components: a low-spin heme indicating a histidinyl-methionyl coordination; a low-spin heme indicating a histidinyl-histidinyl coordination; and a minor high-spin heme component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin hemes, suggesting that for one of the hemes a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the heme-heme interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential heme is high-spin ferrous.