Heme Ligation and Conformational Plasticity in the Isolatedc Domain of Cytochrome cd 1 Nitrite Reductase

• Published in: The Journal of biological chemistry Vol. 276; no. 8; pp. 5846 - 5855
• Main Authors: Steensma, Elles, Gordon, Euan, Öster, Linda M., Ferguson, Stuart J., Hajdu, Janos
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 23.02.2001
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M007345200

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd 1 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 cd 1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd 1 , where the c heme ligands are His 69 -His 17 . An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His 69 -Met 106 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 show 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.