title: Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior.
abstract: Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: A
authors.initials: A
authors.lastname: Koppenhöfer
authors.forename: K L
authors.initials: KL
authors.lastname: Turner
authors.forename: J W
authors.initials: JW
authors.lastname: Allen
authors.forename: S K
authors.initials: SK
authors.lastname: Chapman
authors.forename: S J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D003574:Cytochrome c Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004583:Electrons; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008715:Methionine / Q000378:metabolism; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000201:enzymology*; D011487:Protein Conformation; D013057:Spectrum Analysis; D013696:Temperature; D017186:Titrimetry
chemical_list: D001051:Apoenzymes; D003574:Cytochrome c Group; D003580:Cytochromes; D020035:Holoenzymes; D008024:Ligands; D006418:Heme; D006639:Histidine; C019315:cytochrome cd1; D008715:Methionine; D009572:Nitrite Reductases