Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Evidence for a Single, Catalytically Active, Cytochrome c Binding Domain

Abstract

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.

Binding of Yeast Cytochrome c to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome c Peroxidase: Isothermal Titration Calorimetry

“…The E201K mutation significantly alters the spectroscopic and catalytic properties of the enzyme. 18 The catalytic activity of E201K is very small. In 0.10 M ionic strength potassium phosphate buffer, pH 7.5, the initial velocities for both rCcP and the E210K mutant follow simple Michaelis−Menten kinetics as a function of the yCc concentration.…”

“…Previously we have found that D241K has a UV−vis spectrum characteristic of a predominantly six-coordinate heme at pH 6, it has less than 5% of wildBiochemistry Article DOI: 10.1021/acs.biochem.5b00686 Biochemistry XXXX, XXX, XXX−XXX type catalytic activity for oxidation of yCc 2+ , and CcP Compound I formation is not detected when the mutant reacts with hydrogen peroxide. 18 Further characterization of the D241K mutant shows that its room temperature CD spectrum is similar to that of rCcP, Figure S89, but that its thermal denaturation isotherm is distinctly different, Figure S90. At pH 6.0, in 0.100 M ionic strength potassium phosphate buffer, rCcP denatures in a single, cooperative transition with a midpoint temperature, T m , of 59 ± 1°C and a breadth of 5.6°C (10% to 90% denaturation).…”

“…17,18 Interestingly, only 5 of 46 chargereversal mutants significantly increased the Michaelis constant in the steady-state kinetic studies. Four of the mutants, R31E, D34K, E118K, and E290K occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex between yCc and CcP.…”

Enzymatic Mechanism of Leishmania major Peroxidase and the Critical Role of Specific Ionic Interactions

“…15 The most dramatic effect in yeast CcP is with charge-reversal mutants that create electrostatic clashes at the interface. 39 CcP relies more on nonpolar interactions, while LmP relies on the specific ion pairing between Asp211 in LmP and Arg24 in LmCytc. Despite such large differences, both enzymes conduct the peroxidation of Cytc at similar rates, and the structures of the complexes are remarkably similar.…”

Binding Hot Spot in the Weak Protein Complex of Physiological Redox Partners Yeast Cytochrome c and Cytochrome c Peroxidase

“…19 Finally, Nakani et al and Pearl et al have demonstrated in a series of recent studies that CcP has a single catalytically active Cc binding domainthe one observed in the X-ray structure of the complex. [20][21][22] In the crystal, the Cc-CcP interaction is maintained by multiple van der Waals contacts and a single intermolecular hydrogen bond (Cc N70-CcP E290; Fig. 1a).…”