MopA, the Mn Oxidizing Protein From Erythrobacter sp. SD-21, Requires Heme and NAD+ for Mn(II) Oxidation

Abstract

Bacterial manganese (Mn) oxidation is catalyzed by a diverse group of microbes and can affect the fate of other elements in the environment. Yet, we understand little about the enzymes that catalyze this reaction. The Mn oxidizing protein MopA, from Erythrobacter sp. strain SD-21, is a heme peroxidase capable of Mn(II) oxidation. Unlike Mn oxidizing multicopper oxidase enzymes, an understanding of MopA is very limited. Sequence analysis indicates that MopA contains an N-terminal heme peroxidase domain and a C-terminal calcium binding domain. Heterologous expression and nickel affinity chromatography purification of the N-terminal peroxidase domain (MopA-hp) from Erythrobacter sp. strain SD-21 led to partial purification. MopA-hp is a heme binding protein that requires heme, NAD⁺, and calcium (Ca²⁺) for activity. Mn oxidation is also stimulated by the presence of pyrroloquinoline quinone. MopA-hp has a K _M for Mn(II) of 154 ± 46 μM and k _cat = 1.6 min^-1. Although oxygen requiring MopA-hp is homologous to peroxidases based on sequence, addition of hydrogen peroxide and hydrogen peroxide scavengers had little effect on Mn oxidation, suggesting this is not the oxidizing agent. These studies provide insight into the mechanism by which MopA oxidizes Mn.

Keywords: Mn; MopA; NAD+; PQQ; heme; lactoperoxidase; manganese; peroxidase cyclooxygenase.

FIGURE 1

SDS–PAGE of NAC-purified MopA-hp. NAC-purified bulk sample was analyzed by trypsin digestion and LC–MS/MS. The proteins identified in this sample are indicated in Table 3. Molecular weight size standards as indicated.

FIGURE 2

Heme interaction with MopA-hp. A maximum absorbance shift (from 398 to 412.5 nm) within the Soret region results from protein–heme binding. Dashed line: 3 μM NAC purified MopA-hp; solid line: 0.3 μM heme: dotted line: 3 μM NAC purified MopA-hp with the addition of 0.3 μM heme.

FIGURE 3

Mn oxidizing activity is dependent upon NAD⁺ concentration. Activity normalized to 0.5 mM NAD⁺ concentration. Error bars represent ± one standard deviation of Mn oxidation assays run in triplicates.

FIGURE 4

NADH oxidation by 200 μM Mn(III), E. coli Rosetta^TM 2 “purified” protein (0.11 mg/ml), and NAC purified MopA-hp (0.19 mg/ml).

FIGURE 5

The role of hydrogen peroxide and superoxide on MopA-hp catalyzed Mn(II) oxidation. (A) Hydrogen peroxide addition did not stimulate Mn(II) oxidation, and high concentrations of hydrogen peroxide inhibited activity. Activity normalized to no hydrogen peroxide added. (B) Superoxide dismutase (SOD), up to 10 μM, has little effect on Mn oxidation. Activity normalized to no SOD added. Error bars represent ± one standard deviation of Mn oxidation assays run in triplicate.