title: Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis.
abstract: Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: V W
authors.initials: VW
authors.lastname: Leesch
authors.forename: J
authors.initials: J
authors.lastname: Bujons
authors.forename: A G
authors.initials: AG
authors.lastname: Mauk
authors.forename: B M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002151:Calorimetry; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D018698:Glutamic Acid / Q000235:genetics; D006736:Horses; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015032:Zinc
chemical_list: D003574:Cytochrome c Group; C050095:zinc cytochrome c; D001224:Aspartic Acid; D018698:Glutamic Acid; D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase; D015032:Zinc; D008239:Lysine