Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ

Abstract

A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ) the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though significantly greater than the expected two equivalents were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high valent iron intermediates cannot be ruled out.

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b

“…Though a full EPR kinetic time course is lacking, the appearance of the S=1/2 species within 0.5 min of mixing and its subsequent decay within 300 s is kinetically consistent with its assignment as an intermediate in the conversion of coproheme to heme b, based on the expected reaction t1/2 = 140 s for heme b formation cited above (7). Moreover, the 14 s half-life for the initial decarboxylation of P2 to yield harderoheme and the circa 300 s lifetime of the radical species overall suggests that the observed EPR signals most likely represent superimposed radical intermediate density from both the decarboxylations of P2 and P4, particularly at the later time points (7). This observation is consistent with prior stopped flow analyses, which showed that the P2 and P4 decarboxylations were similar in rate and not temporally well resolved (8).…”

“…By contrast, prior work showed that approximately 10 eq H2O2 was sufficient to convert the WT/unlabeled enzyme-coproheme complex to -heme b; the small excess of H2O2 was required due to competing side reactions between H2O2 and the protein/heme (7,9). A ferric-harderoheme complex accrues with formation rate constant previously fitted to k = 2.9 min -1 or t1/2 = 14 s, and heme b forms with k = 0.30 min -1 (t1/2 = 140 s (pH 7.4 potassium phosphate, 20 °C) (7).…”

“…A ferric-harderoheme complex accrues with formation rate constant previously fitted to k = 2.9 min -1 or t1/2 = 14 s, and heme b forms with k = 0.30 min -1 (t1/2 = 140 s (pH 7.4 potassium phosphate, 20 °C) (7).…”

“…Each decarboxylation is an oxidation in which a net two electrons and two protons are transferred from the reactive propionate to a molecule of H2O2, yielding 2H2O, CO2, and a new vinyl group (7,8). H2O2 activation at the open coordination position on the substrate iron (distal pocket) could generate any of a number of well-known reactive species, including a ferric-hydroperoxy [Fe(III)-OOH], ferryl porphyrin π-cation radical [Fe(IV)=O (por+•), compound I], or ferryl complex [Fe(IV)=O or Fe(IV)-OH compound II].…”

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

“…Recently, Dailey and co-workers proposed a novel haeme biosynthesis pathway for Gram-positive bacteria in which HemQ acts as a coprohaeme decarboxylase to yield protohaeme; in this reaction, coprohaeme has to bind to HemQ, react with presumably H 2 O 2 to release two CO 2 molecules to yield and release haeme b. This finding is also supported by other studies [7][8][9][10]. Nevertheless, the mechanism of this reaction is unknown.…”