Van Wonderen JH, Knight C, Oganesyan VS, George SJ, Zumft WG, Cheesman MR (2007)

Activation of the cytochrome cd₁ nitrite reductase from Paracoccus pantotrophus: reaction of oxidised enzyme with substrate drives a ligand switch at heme c.

J Biol Chem., 282, 28207-28215 (2007).

Cytochromes cd₁ are dimeric bacterial nitrite reductases which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d₁ dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidised enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr25 dissociates from the distal side of heme d₁ and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c and it is believed that Paracoccus pantotrophus cytochrome cd₁ is unreactive towards substrate without reductive activation. However, we report here that the oxidised enzyme will react with nitrite to yield a novel species in which heme d₁ is EPR-silent. Magnetic circular dichroism studies indicate that heme d₁ is low-spin FeIII but EPR-silent as a result of spin-coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at FeIII heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity towards nitrite is also observed for oxidised cytochrome cd₁ from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent FeIII heme d₁ species in nitrite reduction.