How Covalent Heme to Protein Bonds Influence the Formation and Reactivity of Redox Intermediates of a Bacterial Peroxidase

Markus Auer,Andrea Nicolussi,Georg Schütz,Paul G Furtmüller,Christian Obinger

doi:10.1074/jbc.m114.595157

Markus Auer, Andrea Nicolussi + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m114.595157

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Abstract

The most striking feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO) is the existence of covalent bonds between the prosthetic group and the protein, which has a strong impact on their (electronic) structure and biophysical and chemical properties. Recently, a novel bacterial heme peroxidase with high structural and functional similarities to LPO was described. Being released from Escherichia coli, it contains mainly heme b, which can be autocatalytically modified and covalently bound to the protein by incubation with hydrogen peroxide. In the present study, we investigated the reactivity of these two forms in their ferric, compound I and compound II state in a multi-mixing stopped-flow study. Upon heme modification, the reactions between the ferric proteins with cyanide or H2O2 were accelerated. Moreover, apparent bimolecular rate constants of the reaction of compound I with iodide, thiocyanate, bromide, and tyrosine increased significantly and became similar to LPO. Kinetic data are discussed and compared with known structure-function relationships of the mammalian peroxidases LPO and myeloperoxidase.

Highlights

The impact of autocatalytically formed covalent heme to protein bonds on formation and reactivity of redox intermediates of a peroxidase was analyzed
It was reported that the recombinant protein purified from E. coli was a mixture of two species with ϳ80% containing heme b and ϳ20% having the prosthetic group covalently bound to the protein via two ester linkages similar to LPO (Fig. 1D) (13)
We report the kinetics of Reactions 1– 6 of both LspPOX and LspPOX (H2O2) and demonstrate the significant impact of this posttranslational modifications (PTMs) on the individual reaction steps

Summary

Background

The impact of autocatalytically formed covalent heme to protein bonds on formation and reactivity of redox intermediates of a peroxidase was analyzed. In MPO, the heme ring features a bow-shaped structure with less symmetry and a considerable out-of-plane location of the high-spin iron ion (3–5) Formation of these covalent heme to protein bonds has been proposed to occur autocatalytically (7–9) and has a deep impact on the biochemical and biophysical properties of these mammalian peroxidases (2, 10, 11). It was reported that the recombinant protein purified from E. coli was a mixture of two species with ϳ80% containing heme b (see Fig. 1B) and ϳ20% having the prosthetic group covalently bound to the protein via two ester linkages similar to LPO (Fig. 1D) (13) Most interestingly, this ratio shifted significantly when the recombinant protein was incubated with hydrogen peroxide. The reported apparent bimolecular rate constants are compared with those known from the mammalian counterparts and discussed with respect to the known structures of MPO (3–5) and LPO (6)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION