Abstract
Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of −205 ± 3 mV for LmHemQ and −207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 104 M–1 s–1). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria.
Highlights
HemQ is an enzyme involved in the late stages of the heme biosynthetic pathway of monoderm (Gram-positive) bacteria.[1−3] It catalyzes the decarboxylation of iron coproporphyrin III to yield heme b via an unusual peroxidedependent reaction that is poorly understood.[4]
DyPs are versatile peroxidases, capable of performing hydrogen peroxide-dependent one-electron oxidations of various aromatic compounds.[10,11]. In both chlorite dismutases (Cld) and DyPs, a heme b is tightly bound to the protein by a proximal histidine, which is part of an extended H-bonding network.[5,12−16] In addition, Clds have a catalytic distal arginine,[5,17] which is important for stabilization of the transiently produced hypochlorite during the reaction.[18,19]
On the basis of structural and sequence alignments, a glutamine is present in HemQs from Firmicutes (Q187) and an alanine in Actinobacteria at the respective position of the catalytic arginine in Clds or DyPs
Summary
HemQ is an enzyme involved in the late stages of the heme biosynthetic pathway of monoderm (Gram-positive) bacteria.[1−3] It catalyzes the decarboxylation of iron coproporphyrin III (coproheme) to yield heme b via an unusual peroxidedependent reaction that is poorly understood.[4]. Dailey and co-workers discovered that HemQ is an essential enzyme in the heme biosynthesis of Firmicutes and Actinobacteria and identified coproheme as its substrate.[1,24] Coproheme has four propionate groups located at positions 2, 4, 6, and 7 of the porphyrin ring. Propionates at positions 2 and 4 are decarboxylated by HemQ in a stepwise fashion to form the respective vinyl groups of heme b (Figure 1C).[1] The interactions of coproheme with the protein moiety of HemQ as well as the catalytic reaction mechanism of the decarboxylation reactions are unknown. The equilibrium established by the organisms of iron uptake, heme biosynthesis, heme degradation, and iron release is very sensitive and essential for their viability.[28]
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