The Earth's crust hosts a subsurface, dark, and oligotrophic biosphere that is poorly understood in terms of the energy supporting its biomass production and impact on food webs at the Earth's surface. Dark oligotrophic volcanic ecosystems (DOVEs) are good environments for investigations of life in the absence of sunlight as they are poor in organics, rich in chemical reactants and well known for chemical exchange with Earth's surface systems. Ice caves near the summit of Mt. Erebus (Antarctica) offer DOVEs in a polar alpine environment that is starved in organics and with oxygenated hydrothermal circulation in highly reducing host rock. We surveyed the microbial communities using PCR, cloning, sequencing and analysis of the small subunit (16S) ribosomal and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO) genes in sediment samples from three different caves, two that are completely dark and one that receives snow-filtered sunlight seasonally. The microbial communities in all three caves are composed primarily of Bacteria and fungi; Archaea were not detected. The bacterial communities from these ice caves display low phylogenetic diversity, but with a remarkable diversity of RubisCO genes including new deeply branching Form I clades, implicating the Calvin-Benson-Bassham (CBB) cycle as a pathway of CO2 fixation. The microbial communities in one of the dark caves, Warren Cave, which has a remarkably low phylogenetic diversity, were analyzed in more detail to gain a possible perspective on the energetic basis of the microbial ecosystem in the cave. Atmospheric carbon (CO2 and CO), including from volcanic emissions, likely supplies carbon and/or some of the energy requirements of chemoautotrophic microbial communities in Warren Cave and probably other Mt. Erebus ice caves. Our work casts a first glimpse at Mt. Erebus ice caves as natural laboratories for exploring carbon, energy and nutrient sources in the subsurface biosphere and the nutritional limits on life.

The multi-copper oxidase (MCO) MnxG from marine Bacillus bacteria plays an essential role in geochemical cycling of manganese by oxidizing Mn²⁺(aq) to form manganese oxide minerals at rates that are three to five orders of magnitude faster than abiotic rates. The MCO MnxG protein is isolated as part of a multi-protein complex, denoted as Mnx, which includes one MnxG unit and a hexamer of MnxE₃F₃ subunit. During the oxidation of Mn²⁺(aq) catalyzed by the Mnx protein complex, an enzyme-bound Mn(III) species was trapped recently in the presence of pyrophosphate (PP) and analyzed using parallel-mode electron paramagnetic resonance (EPR) spectroscopy. Herein, we provide a full analysis of this enzyme-bound Mn(III) intermediate via temperature dependence studies and spectral simulations. This Mnx-bound Mn(III) species is characterized by a hyperfine-coupling value of A(⁵⁵Mn) = 4.2 mT (corresponding to 120 MHz) and a negative zero-field splitting (ZFS) value of D = - 2.0 cm^-1. These magnetic properties suggest that the Mnx-bound Mn(III) species could be either six-coordinate with a ⁵B_1g ground state or square-pyramidal five-coordinate with a ⁵B₁ ground state. In addition, as a control, Mn(III)PP is also analyzed by parallel-mode EPR spectroscopy. It exhibits distinctly different magnetic properties with a hyperfine-coupling value of A(⁵⁵Mn) = 4.8 mT (corresponding to 140 MHz) and a negative ZFS value of D = - 2.5 cm^-1. The different ZFS values suggest differences in ligand environment of Mnx-bound Mn(III) and aqueous Mn(III)PP species. These studies provide further insights into the mechanism of biological Mn²⁺(aq) oxidation.

Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

MnxG, a multicopper oxidase, is an enzyme from the marine Bacillus species, which produces manganese oxide minerals through the aerobic oxidation of dissolved Mn2+ − a key process in global manganese geochemical cycling. When isolated in an active form as a part of the MnxEFG protein complex, the enzymatic activity of MnxG is substantially enhanced by mild oxidative priming. Herein, the mechanism for this effect is probed by using direct current (dc) and Fourier transformed alternating current (ac) voltammetric analysis of the MnxEFG complex and the catalytically inactive MnxEF subunit immobilised on a carbon electrode. Analysis of these ac voltammetric data reveals a significant enhancement in the rate of electron transfer in the Type 2 Cu sites upon oxidative priming of the enzyme, which is attributed to the improved catalytic activity of MnxG in the MnxEFG protein complex.

The bacterial manganese oxidase MnxG of the Mnx protein complex is unique among multicopper oxidases (MCOs) in carrying out a two-electron metal oxidation, converting Mn(II) to MnO₂ nanoparticles. The reaction occurs in two stages: Mn(II) → Mn(III) and Mn(III) → MnO₂. In a companion study , we show that the electron transfer from Mn(II) to the low-potential type 1 Cu of MnxG requires an activation step, likely forming a hydroxide bridge at a dinuclear Mn(II) site. Here we study the second oxidation step, using pyrophosphate (PP) as a Mn(III) trap. PP chelates Mn(III) produced by the enzyme and subsequently allows it to become a substrate for the second stage of the reaction. EPR spectroscopy confirms the presence of Mn(III) bound to the enzyme. The Mn(III) oxidation step does not involve direct electron transfer to the enzyme from Mn(III), which is shown by kinetic measurements to be excluded from the Mn(II) binding site. Instead, Mn(III) is proposed to disproportionate at an adjacent polynuclear site, thereby allowing indirect oxidation to Mn(IV) and recycling of Mn(II). PP plays a multifaceted role, slowing the reaction by complexing both Mn(II) and Mn(III) in solution, and also inhibiting catalysis, likely through binding at or near the active site. An overall mechanism for Mnx-catalyzed MnO₂ production from Mn(II) is presented.

The dynamics of manganese solid formation (as MnOx) by the multicopper oxidase (MCO)-containing Mnx protein complex were examined by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (CW) EPR spectra of samples of Mnx, prepared in atmosphere and then reacted with Mn(II) for times ranging from 7 to 600 s, indicate rapid oxidation of the substrate manganese (with two-phase pseudo-first-order kinetics modeled using rate coefficients of: k(1obs) = 0.205 ± 0.001 s(-1) and k(2obs) = 0.019 ± 0.001 s(-1)). This process occurs on approximately the same time scale as in vitro solid MnOx formation when there is a large excess of Mn(II). We also found CW and pulse EPR spectroscopic evidence for at least three classes of Mn(II)-containing species in the reaction mixtures: (i) aqueous Mn(II), (ii) a specifically bound mononuclear Mn(II) ion coordinated to the Mnx complex by one nitrogenous ligand, and (iii) a weakly exchange-coupled dimeric Mn(II) species. These findings provide new insights into the molecular mechanism of manganese mineralization.

The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO₂ biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu²⁺, the electron acceptor. Indeed the type 1 Cu²⁺ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO₂ formation from MnO₂ nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pK_a > 8.6 deprotonation, which is assigned to Mn(II)-bound H₂O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pK_a 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH)₂Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O)₂Mn(IV) or an oligomer, which subsequently nucleates MnO₂ nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper .

In a natural geochemical cycle, manganese-oxide minerals (MnO_x ) are principally formed through a microbial process, where a putative multicopper oxidase MnxG plays an essential role. Recent success in isolating the approximately 230 kDa, enzymatically active MnxEFG protein complex, has advanced our understanding of biogenic MnO_x mineralization. Here, the kinetics of MnO_x formation catalyzed by MnxEFG are examined using a quartz crystal microbalance (QCM), and the first electrochemical characterization of the MnxEFG complex is reported using Fourier transformed alternating current voltammetry. The voltammetric studies undertaken using near-neutral solutions (pH 7.8) establish the apparent reversible potentials for the Type 2 Cu sites in MnxEFG immobilized on a carboxy-terminated monolayer to be in the range 0.36-0.40 V versus a normal hydrogen electrode. Oxidative priming of the MnxEFG protein complex substantially enhances the enzymatic activity, as found by in situ electrochemical QCM analysis. The biogeochemical significance of this enzyme is clear, although the role of an oxidative priming of catalytic activity might be either an evolutionary advantage or an ancient relic of primordial existence.

Manganese-oxide minerals (MnO_x) are widely distributed over the Earth's surface, and their geochemical cycling is globally important. A multicopper oxidase (MCO) MnxG protein from marine Bacillus bacteria plays an essential role in producing MnO_x minerals by oxidizing Mn²⁺(aq) at rates that are 3 to 5 orders of magnitude faster than abiotic rates. The MnxG protein is isolated as part of a multiprotein complex denoted as "Mnx" that includes accessory protein subunits MnxE and MnxF, with an estimated stoichiometry of MnxE₃F₃G and corresponding molecular weight of ≈211 kDa. Herein, we report successful expression and isolation of the MCO MnxG protein without the E₃F₃ hexamer. This isolated MnxG shows activity for Mn²⁺(aq) oxidation to form manganese oxides. The complement of paramagnetic Cu(II) ions in the Mnx protein complex was examined by electron paramagnetic resonance (EPR) spectroscopy. Two distinct classes of type 2 Cu sites were detected. One class of Cu(II) site (denoted as T2Cu-A), located in the MnxG subunit, is identified by the magnetic parameters g_∥ = 2.320 and A_∥ = 510 MHz. The other class of Cu(II) sites (denoted as T2Cu-B) is characterized by g_∥ = 2.210 and A_∥ = 615 MHz and resides in the putative hexameric MnxE₃F₃ subunit. These different magnetic properties correlate with the differences in the reduction potentials of the respective Cu(II) centers. These studies provide new insights into the molecular mechanism of manganese biomineralization.

Influence of the conditions for aerobic oxidation of Mn2+(aq) catalysed by the MnxEFG protein complex on the morphology, structure and reactivity of the resulting biogenic manganese oxides (MnO_x ) is explored. Physical characterisation of MnO_x includes scanning and transmission electron microscopy, and X-ray photoelectron and K-edge Mn, Fe X-ray absorption spectroscopy. This characterisation reveals that the MnO_x materials share the structural features of birnessite, yet differ in the degree of structural disorder. Importantly, these biogenic products exhibit strikingly different morphologies that can be easily controlled. Changing the substrate-to-protein ratio produces MnO_x either as nm-thin sheets, or rods with diameters below 20 nm, or a combination of the two. Mineralisation in solutions that contain Fe2+(aq) makes solids with significant disorder in the structure, while the presence of Ca2+(aq) facilitates formation of more ordered materials. The (photo)oxidation and (photo)electrocatalytic capacity of the MnO_x minerals is examined and correlated with their structural properties.