Crystal structure of the Leishmania major peroxidase–cytochrome c complex

Abstract

The causative agent of leishmaniasis is the protozoan parasite Leishmania major . Part of the host protective mechanism is the production of reactive oxygen species including hydrogen peroxide. In response, L. major produces a peroxidase, L. major peroxidase (LmP), that helps to protect the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome c . We have determined the crystal structure of LmP in a complex with its substrate, L. major cytochrome c (LmCytc) to 1.84 Å, and compared the structure to its close homolog, the yeast cytochrome c peroxidase–cytochrome c complex. The binding interface between LmP and LmCytc has one strong and one weak ionic interaction that the yeast system lacks. The differences between the steady-state kinetics correlate well with the Lm redox pair being more dependent on ionic interactions, whereas the yeast redox pair depends more on nonpolar interactions. Mutagenesis studies confirm that the ion pairs at the intermolecular interface are important to both k cat and K M . Despite these differences, the electron transfer path, with respect to the distance between hemes, along the polypeptide chain is exactly the same in both redox systems. A potentially important difference, however, is the side chains involved. LmP has more polar groups (Asp and His) along the pathway compared with the nonpolar groups (Leu and Ala) in the yeast system, and as a result, the electrostatic environment along the presumed electron transfer path is substantially different.

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

“…21 The LmP D211R mutant exhibited similar changes in activity (Figure 4 and Table 2). These results show that either changing electrostatic complementarity at the center of the complex or disrupting a specific ion pair is responsible for the loss of activity.…”

“…16–18 Like yeast CcP, the physiological redox partner for LmP is Cytc, and LmP also forms a stable Trp radical in CpdI. 19 In addition to the crystal structures of LmP and L. major Cytc (LmCytc), 20 the structure of the LmP–LmCytc complex is also known 21 and is very similar to that of the yeast CcP–yCytc complex (Figure 1). …”

“…The role of this ion pair was initially probed by site-directed mutagenesis and steady-state Cytc oxidation assays, which revealed that replacing Arg24 with Ala increases K m by 2-fold and decreases k cat by 30-fold. 21 A decrease in affinity was expected, but such a large decrease in k cat was not, because at saturating levels of LmCytc, k cat might be expected to approach wild-type levels.…”

“…In addition, the steady-state kinetics of LmP are comparatively simple and most consistent with a single binding site for LmCytc, while with yeast CcP, it now is well-established that there is a second site that is populated at low ionic strengths. 15,23 Furthermore, while the complex that forms between CcP and yCytc does not have specific ionic interactions at the interface, 24 the LmP–LmCytc complex depends on specific electrostatic interactions, 21 including a key ionic interaction between LmP Asp211 and LmCytc Arg24, located at the binding interface. The role of this ion pair was initially probed by site-directed mutagenesis and steady-state Cytc oxidation assays, which revealed that replacing Arg24 with Ala increases K m by 2-fold and decreases k cat by 30-fold.…”

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

“…For the experimental work, we have used Leishmania major peroxidase (LmP) rather than yeast CCP. 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).…”