Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd(1) nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytoc hrome ed, nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His(69)-Met(106), and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome ed, nitrite reductas e. In the oxidized state, however, the structure of the isolated c domain i s different from the structure seen in oxidized crystals of intact cytochro me ed,, where the c heme Ligands are His(69)-His(17). An equilibrium mixtur e of heme Ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met(106) has Left the heme iron. Melting studies s how that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1. 2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d(1) heme in guiding His(17) to the c heme in the oxidized holoenzyme is discussed.