From chlorite dismutase towards HemQ–the role of the proximal H-bonding network in haeme binding

Abstract

SynopsisChlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from 'Candidatus Nitrospira defluvii' (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV-vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure-function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria.

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ)

“…Points used for these lines are tabulated in Table S1 of reference 43. Data for Da Clds and Nd Clds are from references 43 and 44, respectively.…”

The HemQ coprohaem decarboxylase generates reactive oxygen species: implications for the evolution of classical haem biosynthesis

“…HemQ enzymes have recently been reported to lack a hydrogen-bonding network in the proximal haem-binding pocket that is present in closely related chlorite dismutase enzymes [17,18]. Such features are common in peroxidase-like enzymes that induce imidazolate character upon the axial histidine ligand that is important in catalysing the O–O bond cleavage reaction of haem-bound peroxides via the well-known ‘push–pull’ model [36] (to stabilise higher oxidation states of the haem iron during the peroxidase catalytic cycle).…”

“…Since HemQ has recently been reported to proceed via a peroxide-dependent reaction [19] and the current data support the generation of superoxide by HemQ, it was of interest to revisit the structural components of the haem-binding cleft. Furthermore, previous structural analyses on HemQ-type enzymes have overlooked the members of this family that have been kinetically characterised and verified to be bona fide HemQ enzymes [17]. Hence, structural models for HemQ enzymes from S. aureus , P. acnes , and M. tuberculosis were generated using the RaptorX server [31,32], and the haem cofactor from the closely related chlorite dismutase from Candidatus Nitrospira defluvii (PDB code 3NN1 [34], Figure 7H) was overlaid (Figure 7A–C).…”

“…However, the previously reported rate constant for the peroxide-dependent evolution of protohaem of 1.6 min −1 is very low [16], which is consistent with a peroxidase-like active site for HemQ that lacks key features, such as a distal pocket arginine (that would stabilise a developing negative charge on the leaving group) and a proximal pocket hydrogen-bonding network (that would give the axial histidine imidazolate character to stabilise higher oxidation states of the iron during the peroxidase catalytic cycle). Previous structural analyses have focused on HemQ family members that lack biochemical confirmation as Fe-coproporphyrin III decarboxylases [17,18], so it was of interest to investigate HemQ enzymes from S. aureus , P. acnes , and M. tuberculosis , which have all been experimentally validated as HemQ enzymes. The structural modelling studies here (Figure 7) suggest that imidazolate character on the axial histidine is not necessary for catalysis, although some HemQ enzymes do possess hydrogen-bonding residues that could potentially polarise this residue.…”

“…S. aureus hemQ mutants were then shown to accumulate coproporphyrin III, and the HemQ enzyme was reported to lyse haem in the presence of peroxide/chlorite [15]. Structural analyses then confirmed the HemQ family of proteins to be distinct from the original assignment to the family of chlorite dismutase enzymes [16]; these differences were defined by variations in conserved proximal/distal pocket residues [17]. However, an overlay of chlorite dismutase coordinates with the crystal structure of apo-HemQ from Listeria monocytogenes revealed the chlorite dismutase haem docks neatly in the HemQ active site cleft [18].…”