title: Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase.
abstract: 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 His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 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 cd1, where the c heme ligands are His69-His17. An equilibrium mixture 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 Met106 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 d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.
authors.affiliation: Department of Biochemistry, Uppsala University, Box 576, 75123 Uppsala, Sweden. elles@xray.bmc.uu.se
authors.forename: E
authors.initials: E
authors.lastname: Steensma
authors.forename: E
authors.initials: E
authors.lastname: Gordon
authors.forename: L M
authors.initials: LM
authors.lastname: Oster
authors.forename: S J
authors.initials: SJ
authors.lastname: Ferguson
authors.forename: J
authors.initials: J
authors.lastname: Hajdu
mesh_terms: D003580:Cytochromes; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D008024:Ligands; D008958:Models, Molecular; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011522:Protons; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry
chemical_list: D003580:Cytochromes; D006420:Hemeproteins; D008024:Ligands; D010446:Peptide Fragments; D011522:Protons; D012996:Solutions; D006418:Heme; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV