Oxygen Generation via Water Splitting by a Novel Biogenic Metal Ion-Binding Compound

Abstract

Methanobactins (MBs) are small (<1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu²⁺ reduced to Cu⁺. Other metal ions, however, are bound but not reduced, e.g., K⁺. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H₂¹⁸O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au³⁺, Cu², or Ag⁺, ^18,18O₂ and free protons were released. No ^18,18O₂ production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K⁺ or Mo²⁺ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe³⁺ with an N₂S₂ coordination and will also reduce Fe³⁺ to Fe²⁺. Iron reduction was also found to be coupled to the oxidation of 2H₂O and the generation of O₂. MB-SB2 will also couple Hg²⁺, Ni²⁺, and Co²⁺ reduction to the oxidation of 2H₂O and the generation of O₂, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions. To determine if the O₂ generated during metal ion reduction by MB could be coupled to methane oxidation, ¹³CH₄ oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate that O₂ generation from metal ion reduction by MB-OB3b can support methane oxidation. IMPORTANCE The discovery that MB will couple the oxidation of H₂O to metal ion reduction and the release of O₂ suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.

Keywords: aerobic methane oxidation; chalkophore; gold nanoparticle; methanobactin; methanotroph; water oxidation.

FIG 1

Gold X-ray photoelectric spectra of MS-SB2 at a gold/MB-SB2 molar ratio of 14 to 1 after a 30-min incubation (red circles) (A) and at a gold/MB-SB2 molar ratio of 19 to 1 after a 30-min incubation (B). Experimental results (circles) were fit with CASA XPS software to four Gaussian/Lorentzian curves, using two peaks for Au³⁺ (orange curves) and two peaks for Au⁰ (blue curves). Gold 4f core electrons are spin-orbit split as 4f_7/2 and 4f_5/2, with a splitting of 3.7 eV and area ratio of 4:3, so that only two peaks are independently fit: the 4f_7/2 peaks for Au³⁺ and Au⁰. The 4f_5/2 peaks’ positions and areas are determined by spin-orbit splitting; these parameters and the peak widths are fixed in the fitting program. The background used was of a Shirley type.

FIG 2

(A) Kinetics of Au binding by MB-SB2 at 4°C. (A) Rate of HAuCl₄ binding to the imidazolone (△) and oxazolone (○) rings of MB-SB2 at 4°C as measured from the absorbance changes at 386 nm and 341 nm, respectively. The rates for Au binding were >2,000 s⁻¹ and were set at 2,000 s⁻¹ in the figure. (B) Emission at 429 nm from SB2-MB following excitation at 341 nm after the addition of 0 (light blue), 0.25 (orange), 0.5 (gray), 0.75 (yellow), or 2.25 (dark blue) HAuCl₄ per MB-SB2. a.u., arbitrary units.

FIG 3

(A) pH changes following the addition of HAuCl₄ to aqueous solutions (gray triangles) or an aqueous solution of 40 μM MB-SB2 (yellow circles). (B) pH changes following the addition of CuCl₂ to aqueous solutions (gray triangles) or an aqueous solution of 40 μM MB-SB2 (blue circles). (C) pH changes following the addition of KCl to aqueous solutions (gray triangles) or an aqueous solution of 40 μM MB-SB2 (black circles).

FIG 4

Mass spectra of the headspace gas of a reaction mixture containing 2 mM MB-SB2 in 97% H₂¹⁸O (A) and following the addition of 20 mM HAuCl₄ (B), 20 mM CuCl₂ (C), 10 mM CuCl₂ (D), 20 mM KCl (E), 20 mM AgF (F), 20 mM FeCl₃ (G), 20 mM HgCl₂ (H), 20 mM NiCl₂ (I), and 20 mM CoCl₂ (J).

FIG 5

^18,18O₂ concentration in the headspace of a reaction mixture containing 2 mM MB-SB2 plus 20 mM HAuCl₄ (yellow triangles) or 20 mM CuCl₂ (light blue circles) in 97% H₂¹⁸O and following the addition of 7.3 mM catalase.

FIG 6

(A and B) Iron reductase activities of MB-SB2 (A) and MB-OB3b (B). The absorption change at 562 nm of reaction mixtures containing 1 mM ferrozine plus 10 mM FeCl₃, 1 mM ferrozine plus 23.4 μM MB-SB2, 1 mM ferrozine plus 10 mM FeCl₃, and either 5.8, 11.6, 17.4, or 23.4 μM MB-SB2 (A) or MB-OB3b (B) was measured. (C) Aqueous 4 M FeCl₃ solution and 4 M FeCl₃ solution plus 20 mM MB-SB2 4 h after the addition of MB-SB2.

FIG 7

Mass spectra of the headspace gas of a reaction mixture containing 2 mM MB-OB3b in 97% H₂¹⁸O (A) and following the addition of 20 mM HAuCl₄ (B), 20 mM CuCl₂ (C), and 20 mM AgF (D).

FIG 8

¹³CO₂ production by the M. trichosporium OB3b wild type (WT), the WT plus 25 μM CuCl₂, the WT plus 5 μM MB-OB3b, and the WT plus 25 μM CuCl₂ and 5 μM MB-OB3b incubated in an anaerobic glove box for 3 days.