Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs

https://doi.org/10.1016/j.jinorgbio.2008.02.003Get rights and content

Abstract

Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O2. Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O2 to O2-. Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of O2- to H2O2, and the reductant dependent reduction of H2O2 to H2O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.

Introduction

Methanobactin (mb) is a copper-binding chromopeptide found in both the extracellular and membrane fractions of many if not all aerobic methane oxidizing bacteria, or methanotrophs [1], [2], [3], [4], [5], [6], [7]. The crystal structure of copper-containing mb (Cu-mb) from Methylosinus trichosporium OB3b showed the molecule represented a new class of metal binding compounds with a primary sequence of N-2-isopropylester-(4-thiocarbonyl-5-hydroxy imidazolate)-Gly-Ser-Cys-Tyr-pyrrolidine-(4-hydroxy-5-thiocarbonyl-imidazolate)-Ser-Cys-Met [6]. Copper coordination was also unique with a dual S, and N coordination by 4-thiocarbonyl-5-hydroxy imidazolate (THI) and 4-hydroxy-5-thiocarbonyl imidazolate (HTI) [6]. Recent studies have also suggested that mb is a dynamic molecule in solution and appears to initially bind Cu(II) as a multimer, probably a tetramer, via THI and Tyr [2], [3]. This initial binding is followed by a reduction to Cu(I) then by coordination to HTI. Studies on the metal binding as well as on the solution and thermodynamic properties of mb from Ms. trichosporium OB3b suggest the physiological function of mb is that of a copper siderophore or chalkophore [2], [3], [4], [5], [6], [7], [8], [9].

In addition to the extracellular fraction, Cu-mb is also found in the cell membrane fraction [7]. In fact, Cu-mb was initially identified in association with the membrane-associated or particulate methane monooxygenase (pMMO) and was originally proposed as a cofactor of the hydroxylase component of the pMMO (pMMO-H) [1], [7], [10]. In this model, the pMMO was a complex composed of three polypeptides (pmoA, pmoB, and pmoC), i.e., the hydroxylase (pMMO-H) component plus 5–8 Cu-mb [1], [7], [10], [11]. Subsequent studies in other laboratories have reported active preparations of pMMO-H with no evidence of Cu-mb [12], [13], [14], [15], [16], [17], [18], [19]. The reported activities of pMMO-H in the absence of Cu-mb, however, were low, only 2–25% of the reported activities for pMMO-H isolated with Cu-mb.

In addition to co-purification, the culture conditions used to stabilize cell free pMMO activity also suggest an association between Cu-mb and pMMO [1], [10]. The high copper conditions used to stabilize the pMMO results in increased concentrations of membrane-associated Cu-mb. Cu-mb has also been shown to increase electron flow to the type II Cu(II) centers of pMMO-H and to have superoxide dismutase activity suggesting secondary roles of Cu-mb in methane catalysis by the pMMO [1], [10]. To examine the potential of mb in pMMO-H stabilization or in electron flow to pMMO-H, the oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of mb was examined and compared to the effect of mb on pMMO-H.

Section snippets

Organisms, culture conditions and isolation of membrane fraction

Culture conditions for the isolation of mb from the spent media of M. trichosporium OB3b were described previously [3]. Similar cultivation conditions were used for the isolation of mb from Methylococcus capsulatus Bath, and from Methylomicrobium album BG8. Methanobactin (mb) was prepared from the spent medium of Ms. trichosporium OB3b, Mc. capsulatus Bath and Mm. album BG8 as previously described for Ms. trichosporium OB3b [3], [10].

For the isolation of the washed membrane fraction and pMMO

Extracellular concentration of mb

The highest yields of mb in the spent media of all three methanotrophs were obtained from nitrate minimal salts media (NMS) amended with 0.2 μM CuSO4, but the yields varied with Ms. trichosporium OB3b showing the highest concentrations (35–60 mg l−1) followed by Mc. capsulatus Bath (18–24 mg l−1) and Mm. album BG8 (1–10 mg l−1).

Superoxide dismutase-like activity

Cu-mb has been shown to have superoxide dismutase-like (SOD) activity [10]. To determine if the stimulatory and/or inhibitory effects of mb on pMMO activity were related to

Abbreviations

    Cu-mb

    methanobactin copper complex

    HPR

    hydrogen peroxide reductase

    HTI

    4-hydroxy-5-thiobarbonyl imidazolate

    Mb

    methanobactin

    MMO

    methane monooxygenase

    NBT

    nitroblue tetrazolium

    P

    Pearson correlation

    pMMO

    membrane-associated or particulate methane monooxygenase

    pMMO-H

    hydroxylase component of the membrane-associated methane monooxygenase

    PMS

    phenazine methosulfate

    R

    significance (2-tailed)

    sMMO

    soluble methane monooxygenase

    SOD

    superoxide dismutase

    THI

    4-thiocarbonyl-5-hydroxy imidazolate

    XPS

    X-ray photoelectric spectroscopy

Acknowledgements

This work was supported by the Department of Energy Grants 96ER20237 to A.D.S. and W.A. and DE-FC26-05NT42431 to J.D.S. and by a grant from the Battelle BioScience Alliance of Iowa to A.D.S.

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