Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry.
Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C. The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3-. CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined. Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1). The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.
Jacobson
Timothy
T
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
Williamson
Joy
J
Wasilewski
Anthony
A
Felesik
Janet
J
Vitello
Lidia B
LB
Erman
James E
JE
D000818:Animals; D001386:Azides / Q000378:metabolism*; D002021:Buffers; D002151:Calorimetry / Q000379:methods; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008786:Metmyoglobin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry / Q000379:methods; D013816:Thermodynamics; D017186:Titrimetry
D001386:Azides; D002021:Buffers; D008786:Metmyoglobin; D003578:Cytochrome-c Peroxidase