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

Abstract

Leishmania major peroxidase (LmP) is very similar to the well-known yeast cytochrome c peroxidase (CcP). Both enzymes catalyze the peroxidation of cytochrome c. Like CcP, LmP reacts with H2O2 to form Compound I, which consists of a ferryl heme and a Trp radical, FeIV= O;Trp•+. Cytochrome c (Cytc) reduces the Trp radical to give Compound II, FeIV= O;Trp, which is followed by an intramolecular electron transfer to give FeIII–OH;Trp•+, and in the last step, Cytc reduces the Trp radical. In this study, we have used steady-state and single-turnover kinetics to improve our understanding of the overall mechanism of LmP catalysis. While the activity of CcP greatly increases with ionic strength, the kcat for LmP remains relatively constant at all ionic strengths tested. Therefore, unlike CcP, where dissociation of oxidized Cytc is limiting at low ionic strengths, association/dissociation reactions are not limiting at any ionic strength in LmP. We conclude that in LmP, the intramolecular electron transfer reaction, FeIV= O;Trp to FeIII–OH;Trp•+, is limiting at all ionic strengths. Unlike CcP, LmP depends on key intermolecular ion pairs to form the electron transfer competent complex. Mutating these sites causes the initial rate of association to decrease by 2 orders of magnitude and a substantial decrease in kcat. The drop in kcat is due to a switch in the rate-limiting step of the mutants from intramolecular electron transfer to the rate of association in forming the LmP–LmCytc complex. These studies show that while LmP and CcP form very similar complexes and exhibit similar activities, they substantially differ in how their activity changes as a function of ionic strength. This difference is primarily due to the heavy reliance of LmP on highly specific intermolecular ion pairs, while CcP relies mainly on nonpolar interactions.

Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer

“…As noted in the Introduction, LmP is also a CCP and forms the same stable Trp radical (17), and the crystal structure of the LmP-LmCytc complex (16) is very similar to the CCPCytc complex (27). LmP, however, exhibits simpler kinetics because k cat for LmP is relatively insensitive to ionic strength (14), whereas k cat for CCP increases substantially with ionic strength (25). This increase is generally thought to be the result of a change in rate-limiting step in CCP, where product dissociation is limiting at low ionic strength and reduction of CmpII is limiting at high ionic strength (25,26).…”

“…1C, protonation of the ferryl O atom during reduction of CmpII requires transfer of the His52 proton via ordered solvent. This step is thought to be rate-limiting for CCP at high ionic strength (25,26), whereas for the closely related LmP, this step is limiting at all ionic strengths (14). If CmpII reduction is limiting and solvent plays an important role in proton transfer, as suggested by the CmpI structures, then one might predict a considerable KSIE.…”

“…Crystals ranging between 150 μm and 1 mm in length were harvested onto Hampton-style cryoloops, flash-frozen, and stored in a Stanford Synchrotron Radiation Lightsource (SSRL) cassette. LmP and LmCytc used in KSIE experiments were expressed and purified as previously reported (14).…”

“…The pKa is important, because we recently showed that the proton-coupled electron transfer reduction of CmpII (Fig. 1C) is rate-limiting in peroxidase catalysis (14) and a protonated His52 could directly participate in proton transfer to the ferryl O atom during reduction of Fe(IV)=O. Therefore, we also present computational and kinetic solvent isotope effect (KSIE) experiments that examine the pK a of His52 and address the importance of proton transfer in CmpII reduction.…”

“…We and others have shown that LmP is structurally and functionally very similar to yeast CCP, including the structure of the LmP-cytochrome c (Cytc) complex, stability of CmpI, and formation of the active site Trp radical (15)(16)(17)(18). LmP, however, offers some advantages owing to simplified kinetics because, unlike yeast CCP, LmP has only one binding site for Cytc and k cat is independent of ionic strength (14). As a result, the interpretation of kinetic results is more straightforward with LmP.…”