Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L)

Abstract

Imidazole, 1-methylimidazole and 4-nitroimidazole bind to yeast cytochrome c peroxidase (yCcP) with apparent equilibrium dissociation constants (KDnormalapp) of 3.3 ± 0.4, 0.85 ± 0.11, and ~0.2 M, respectively, at pH 7. This is the weakest imidazole binding to a heme protein reported to date and it is about 120 times weaker than imidazole binding to metmyoglobin. Spectroscopic changes associated with imidazole and 1-methylimidazole binding to yCcP suggest partial ionization of bound imidazole to imidazolate. The pKa for ionization of bound imidazole is estimated to be 7.4 ± 0.2, about 7 units lower than that of free imidazole and about 3 units lower than imidazole bound to metmyoglobin. Equilibrium binding of imidazole to CcP(H52L) is biphasic with low- and high-affinity phases having KDnormalapp values of 9.5 ± 4.5 and 0.13 ± 0.04 M, respectively. CcP(H52L) binding of 1-methylimidazole is monophasic with an affinity similar to those of yCcP and rCcP. Binding of 1-methylimidazole to rCcP is associated with two kinetic phases, the initial binding complete within 10 s, followed by a process that is consistent with 1-methylimidazole binding to a cavity created by movement of Trp-191 from the interior of the protein to the surface. Both the equilibrium binding and kinetics of 1-methylimidazole binding to yCcP are pH dependent. yCcP has a four-fold increase in 1-methylimidazole binding affinity on decreasing the pH from 7.5 to 4.0, an observation that is unique among the many studies on binding of imidazole and imidazole derivatives to heme proteins.

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants

“…The expression system for the recombinant CcP (rCcP) used in this study as well as the protocols for construction, expression and purification of the CcP mutants have been previously described [7,8]. …”

“…Concentration of stock protein solutions were determined spectrophotometrically at pH 6.0 using the following extinction coefficients in the Soret band: yCcP, 98 mM −1 cm −1 at 408 nm; rCcP, 101 mM −1 cm −1 at 408 nm; CcP(triAla), 110 mM −1 cm −1 at 406 nm; CcP(triVal), 76 mM −1 cm −1 at 406 nm; and CcP(triLeu), 93 mM −1 cm −1 at 400 nm [7,8]. …”

“…(See Fig. 1 of the preceding paper [8] for the active site structure of CcP). All three mutants show a 30- to 34-fold increase in the rate of 1-methoxynaphthalene hydroxylation by hydrogen peroxide [9].…”

“…In contrast to the loss of cyanide affinity, the three apolar mutants show a substantial increase in imidazole affinity compared to wild-type CcP. In the preceding paper, we have shown that wild-type yeast cytochrome c peroxidase (yCcP) and a recombinant form of CcP (rCcP) have very weak affinities for imidazole with equilibrium dissociation constants ( K D ) of about 4 M at pH 7 [8]. These are the weakest imidazole binding affinities reported for any heme protein.…”

“…At pH 7, the values of K D1 for the triple mutants range from 83 µM for CcP(triVal) to 0.60 mM for CcP(triLeu), showing increased imidazole binding affinity of 3.8 to 4.7 orders of magnitude. Even the low-affinity binding phases of the triple mutants bind imidazole more strongly than wild-type CcP with K D2 values ranging from 12 to 17 mM at pH 7, some 2.5 orders of magnitude smaller than K D for the CcP/imidazole complex [8]. …”