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 fromcoproheme 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.

Chemistry and Molecular Dynamics Simulations of Heme b‑HemQ and Coproheme-HemQ / Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Oostenbrink, Chris; Obinger, Christian. - In: BIOCHEMISTRY. - ISSN 0006-2960. - STAMPA. - 55:(2016), pp. 5398-5412. [10.1021/acs.biochem.6b00701]

Chemistry and Molecular Dynamics Simulations of Heme b‑HemQ and Coproheme-HemQ

BATTISTUZZI, Gianantonio;
2016

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 fromcoproheme 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.
2016
15-set-2016
55
5398
5412
Chemistry and Molecular Dynamics Simulations of Heme b‑HemQ and Coproheme-HemQ / Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Oostenbrink, Chris; Obinger, Christian. - In: BIOCHEMISTRY. - ISSN 0006-2960. - STAMPA. - 55:(2016), pp. 5398-5412. [10.1021/acs.biochem.6b00701]
Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Oostenbrink, Chris; Obinger, Christian
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1124726
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