Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs
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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2021, International Journal of Mass SpectrometryCitation Excerpt :Methanobactins have the potential as therapeutics in reducing copper toxicity as in Indian childhood cirrhosis, Wilson's disease, and Menkes disease [5–8]. The methanobactin from Methylosinus trichosporium OB3b is the most studied methanobactin (mb-OB3b) and has the structure 1-(N-[mercapto-(5-oxo-2-(3-methylbutanoyl)-oxazol-(Z)-4-ylidene)methyl]-Gly1-Ser2-Cys3-Tyr4)-pyrrolidin-2-yl-(mercapto-[5-oxo-oxazol-(Z)-4-ylidene]methyl)-Ser5-Cys6-Met7 (Fig. 1). [9–18] In mb-OB3b's native form, amino acids and enethiol oxazolone functional groups are present in its sequence and a disulfide bridge is formed between the Cys3 and Cys6 residues [18].