Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla, TriVal, and TriLeu variants

Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 09/2012; 1834(1). DOI: 10.1016/j.bbapap.2012.09.005
Source: PubMed


Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H(2)O(2) compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H(2)O(2) and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H(2)O(2) and HCN while features that inhibit substrate ionization slow the reactions.

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    • "CYP2J2-Mediated Metabolism of Endocannabinoids each substrate tested, rapid mixing and formation of the heme-cyanide complex red-shifted the Soret (Fig. 6A). As shown in Fig. 6B, the difference spectra D (A 444–414nm ) were analyzed as a function of time and yielded an exponential biphasic curve that was comprised of a fast and slow phase (Bidwai et al., 2013). The fast phase corresponds to the rate of cyanide binding to the ferric heme active site. "
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    ABSTRACT: The endocannabinoids anandamide (AEA) and 2 -arachidonoylglycerol (2-AG) are arachidonic acid derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well-known for the metabolism of arachidonic acid (AA) to the biologically active epoxyeicosatrienoic acids (EETs). Herein we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids (EET-EA) whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides (EET-G). Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH dependent reaction with CYP2J2 and cytochrome P450 reductase (CPR). The formation of the predominant AEA and 2-AG epoxides were confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small molecule ligand egress and molecular modeling. The experimental and theoretical data were in agreement which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane bound cardiovascular epoxygenase.
    Full-text · Article · Oct 2014 · Journal of Pharmacology and Experimental Therapeutics
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    • "They were constructed by simultaneously replacing Arg48, Trp51, and His52 with either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). We have previously reported on the reaction of these mutants with hydrogen peroxide and cyanide [16]. As anticipated, the reaction with H2O2 is substantially reduced but the peroxygenase activity is increased by a factor of 34. "
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    Full-text · Article · Jul 2013 · BMC Biochemistry
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    Full-text · Article · Apr 2014 · ChemBioChem
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