Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b

Abstract

A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH dependent, and further decelerated by D2O2 (average KIE = 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme-proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

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

“…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).…”

“…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].…”

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

“…S6). 22 These results suggest that the Mn(III)coproheme binds in the catalytic orientation but is incapable of activating H 2 O 2 and therefore is stable as a solid state complex in the presence of O 2 and ionizing X-ray radiation, conditions under which H 2 O 2 may be spontaneously produced.…”

“…36 Prior work showed that the second order reaction between the decarboxylase-coproheme complex and H 2 O 2 , which leads to the ferric heme b complex, accelerates with pH (~3-fold, p K a = 7.4) and is solvent-isotope dependent (average k (H 2 O): k (D 2 O) = 2.2). 22 Both results suggest the involvement of proton movement in the rate limiting step of the H 2 O 2 reaction. However, pH has a much larger influence over the analogous, acid-base-catalyzed steps in heme peroxidases.…”

“…Some role for the protein is supported by prior studies of the reaction of the coproheme decarboxylase with peracetic acid: an oxygen atom donor that directly converts ferric hemes to Fe(IV)=O(por■+) or Fe(IV)=O(Trp■+) without the proton movement or heterolytic bond cleavage associated with H 2 O 2 . 22 The ferric coproheme decarboxylase reacts rapidly with peracetic acid to form the expected harderoheme intermediate and heme b product. The second order rate constant for the same reaction with H 2 O 2 is 10-fold slower and, in contrast with the peracid reaction, dependent on both pH and solvent isotope (D 2 O).…”