Identification of Proximal and Distal Axial Ligands in Leishmania major Pseudoperoxidase

Abstract

Previous optical and electron paramagnetic resonance (EPR) spectroscopic studies of the newly discovered peroxynitrite scavenging pseudoperoxidase from Leishmania major (LmPP) suggested that ferric LmPP contained a six-coordinate low-spin (6cLS) heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. To identify the axial ligands of LmPP, we exploit a systematic mutational analysis of potential heme ligands. On the basis of UV-visible and EPR spectroscopy, we report that the substitution of the proximal His206 with alanine in LmPP alters the 6cLS to a five-coordinate high spin (5cHS) form at pH 4.0 that has a spectrum characteristic of a Cys-ligated 5cHS derivative. The electronic absorption and EPR analysis of all alanine-substituted Cys and Met single mutants establish that when Cys107 is replaced with alanine, a new species appears that has a spectrum characteristic of a histidine-ligated 5cHS derivative at pH 4.0. Together, these results suggest that His206 and Cys107 act as the proximal and distal axial ligands in ferric LmPP, respectively. However, the electronic properties of reduced wild-type LmPP are similar to those of known 5cHS His-ligated heme proteins at pH 8.8, indicating that the thiolate bond was broken upon reduction. Furthermore, the wild-type protein was only partially reduced at pH 4.0, but the E105L mutant was completely reduced to form a 5cHS ferrous heme. These results imply that the presence of an acidic residue near the distal site may prevent reduction of the heme iron at acidic pH.

Crystal structure and functional analysis of Leishmania major pseudoperoxidase

“…Our crystal structure confirms previous work identifying the LmPP axial ligands using a combination of site-directed mutagenesis and UV/Vis spectrophotometry [19]. The heme is coordinated proximally by His 206 and distally by Cys 107 (Figure 2A).…”

“…UV/Vis spectroscopic studies also showed that the addition of PN causes a shift in the LmPP heme iron from ferric 6-coordinate low spin state to ferric high spin state in the absence of an electron donor. While mutagenesis studies point to His 206 and Cys 107 as the likely proximal and distal axial heme ligands, respectively [19], the absence of a crystal structure has been an obstacle to determining the mechanism of PN detoxification by LmPP. Here, we describe the 1.76-Å crystal structure of LmPP, in addition to kinetic and spectroscopic studies, in order to shed further light on the potential biological function of this intriguing heme protein.…”

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes

“…The His/Cys − coordination found for Heme 1 in the TsdA enzymes is rare and, although the purpose of such ligation is not always clear (39), some examples are suggested to function as redox sensors (40–42) and others as receptors or regulators (43–47). Reported E m values lie in the range −350 to −160 mV (40–43, 47).…”

Effect of temperature, pH and heme ligands on the reduction of Cygb(Fe3+) by ascorbate

“…3B suggest that the increased reduction product Cygb(Fe 2+ ) is likely caused by the increased A 2- concentration. It has demonstrated that increasing pH will stabilize the reduced state of the heme iron as shown in the case of alkaline cystathionine beta-synthase [35] and pseudoperoxidase from Leishmania major (LmPP) [36]. This stabilization may also contribute to the increase in the rate constant of Cygb(Fe 3+ ) reduction by Asc with increasing pH.…”