Spectroscopic and computational investigations of organometallic complexation of group 12 transition metals by methanobactins from Methylocystis sp. SB2

Highlights

• Methylocystis sp. strain SB2 methanobactin forms complexes with Group 12 transition metals.

• Absorbance and fluorescence spectra change with methanobactin-metal stoichiometry and time.

• Hg L3-edge extended x-ray absorption fine structure suggests linear Hg—S coordination.

• Computational results suggest Hg²⁺-methanobactin coordination differs from Zn²⁺ and Cd²⁺

• Fluorescence enhancement observed suggests complexation-hindered isomerization mechanism.

Abstract

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as “methanobactins,” which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for HgS coordination, and TD-DFT suggests a tetrahedral model for Zn²⁺ and Cd²⁺. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.

Introduction

Anthropogenic emissions of greenhouse gases are a central factor in one of the most pivotal environmental issues of the twenty-first century, global climate change. One such greenhouse gas is methane, which is released to the atmosphere by anthropogenic and natural sources. Natural biotic methane production (methanogenesis) occurs primarily through the anaerobic metabolism of carbon dioxide and organic matter by archaea (methanogens). Methane emissions from methanogens are naturally modulated by methane-oxidizing bacteria (methanotrophs), which can metabolize methane as the sole source of carbon and energy [1,2]. The first step in the oxidation of methane to methanol by aerobic methanotrophs involves the enzyme methane monooxygenase (MMO). Two forms of MMO have been characterized: soluble MMO (sMMO), which contains an Fe-based active site and is found in the cytoplasm, and particulate MMO (pMMO), which is membrane-associated and contains Cu [[3], [4], [5], [6], [7]]. Copper is a relatively sparse transition metal, with a natural abundance of 60 ppm in the earth's crust and 0.25 ppb in the ocean [8]. sMMO is typically expressed under Cu-limiting conditions and pMMO at higher Cu concentrations [[9], [10], [11]], and methanotrophic bacteria excrete Cu-binding metallophores (chalkophores) to facilitate Cu acquisition [[12], [13], [14], [15], [16], [17]]. These post-translationally modified peptides (~850–1300 Da) are known as methanobactins and function in a manner analogous to siderophores in the extracellular acquisition of Fe [18,19]. Methanobactins are excreted by methanotrophic bacteria under conditions of Cu deprivation [11]. Although extracellular Cu acquisition is believed to be their primary function, several studies suggest methanobactins may also serve other functions in environmental systems. For example, methanobactins are hypothesized to have a secondary function as signaling molecules within methanotrophic communities [20] and they have been shown to reduce toxicity associated with mercuric mercury (Hg²⁺) in pure culture studies with methanotrophs [21]. Furthermore, recent studies demonstrated that methanobactins facilitate the degradation of highly toxic methylmercury (MeHg) [22], and they also increase the bioavailability of inorganic Hg for methylation by some anaerobic bacteria [23]. Thus, methanobactins may affect net MeHg production in environments where methanotrophs are prevalent.

In addition to their biogeochemical significance, methanobactins are ideal for experimental analysis using optical spectroscopic methods. Unlike most proteins and peptides, methanobactins contain biochemically unusual oxazolone-, imidazolone-, and pyrazine-based chromophores that absorb strongly across the entire UV spectrum and into the visible. The chromophores are directly involved in the complexation of transition metals, so the absorbance and fluorescence spectra of methanobactins are sensitive to the coordination state of the peptide (Fig. 1). Early spectroscopic and isothermal calorimetry experiments demonstrated that methanobactins may form coordination complexes with many early- and mid-transition metals [21,24] and ultimately influence transition metal bioavailability. Similar investigations of methanobactins from Methylosinus trichosporium OB3b (mb-OB3b) and Methylocystis sp. strain SB2 (mb-SB2) have revealed characteristic concentration-dependent changes in spectral characteristics upon binding of Hg²⁺ [21,24] and suggested that both chromophores are involved in the complexation. Furthermore, although methanobactins have a high affinity for binding Cu ions, Hg²⁺ was found to displace Cu²⁺ bound to mb-SB2 [21,24]. However, the specific coordination geometries induced by the complexation of methanobactins with metals are not well understood. Electronic structure calculations in combination with thorough spectroscopic characterization can provide insights into binding geometries and unique molecular properties associated with the binding of different metal ions. The absorbance and fluorescence spectra of methanobactin peptides have not been studied theoretically and the specific correlations between changes in spectroscopic and molecular features have yet to be established. Here, we conducted time-dependent density functional theory (TD-DFT) calculations to describe the geometry and electronic structure of mb-SB2 chromophores, which give rise to characteristic UV–visible absorbance spectra. Furthermore, we collected fluorescence spectra and relate observed emissions to coordination geometries of mb-SB2 complexed with Zn²⁺, Cd²⁺, and Hg²⁺. Finally, the structure of mb-SB2 complexed with Hg²⁺at variable molar ratios was probed with Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy and compared with geometries predicted by TD-DFT calculations. Our data offer molecular-scale insights into the interactions of trace metals with the functional groups present in methanobactins. Understanding these interactions will aid in developing improved speciation models that adequately describe the effect of methanobactins on the biogeochemical cycling of Group 12 transition metals.