A new record of Percursaria percursa (Ulvaceae, Ulvales) on the North Island, New Zealand

The filamentous green alga Percursaria percursa (Ulvaceae, Ulvales) was recorded for the first time on the North Island of New Zealand at mokoroa Estuary, Tauranga Harbour. This species is previously known within New Zealand from only two records, both from the South Island. In Tauranga Harbour, this species was restricted to anoxic estuarine sediments where mangrove forests had been mulched, and mulchate left in situ. Percursaria percursa was found intertwined with Ulva spp. and Rhizoclonium spp. Surveys of other North and South Island estuaries suggest that this alga, although occurring as part of nuisance green algal blooms in Tauranga Harbour, has only colonized human-impacted locations, and has not yet been observed in natural' estuarine ecosystems in New Zealand. As this species was found intertwined with other mat-forming filamentous green algae, it can easily be misidentified in the field, leading to both over- and under-reporting of species occurrence

¹³C NMR Characterization of an Exchange Reaction between CO and CO₂ Catalyzed by Carbon Monoxide Dehydrogenase

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO₂ at a nickel−iron−sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe ¹³C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO₂ in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the ¹³CO NMR resonance; however, the chemical shift of the ¹³CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The ¹³CO line broadening occurs with a rate constant (1080 s⁻¹ at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between ¹³CO and CODH-bound ¹³CO₂ because ¹³CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the ¹³CO₂ line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between ¹²CO and ¹³CO₂ in solution was shown to occur at the same rate as that of CO₂ reduction, which is approximately 750-fold slower than the rate of ¹³CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN⁻) was also probed by ¹³C NMR. A functional C-cluster is not required for ¹³CN⁻ broadening (unlike for ¹³CO), and its exchange rate constant is 30-fold faster than that for ¹³CO. The combined results indicate that the ¹³CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO₂, but not release of CO₂ or protons into the solvent. They also provide strong evidence of a CO₂ binding site and of an internal proton transfer network in CODH. ¹³CN⁻ exchange appears to monitor only movement of CN⁻ between solution and its binding to and release from CODH

Geometric and Electronic Structures of the Ni^I and Methyl−Ni^III Intermediates of Methyl-Coenzyme M Reductase

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni−F₄₃₀ cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the Ni^I active state (MCR_red1) on Me-SCoM to form a Ni^III−methyl intermediate, while computational studies indicate that the first step involves the attack of Ni^I on the sulfur of Me-SCoM, forming a CH₃^• radical and a Ni^II−thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the Ni^I (MCR_red1), Ni^II (MCR_{red1−silent}), and Ni^III−methyl (MCR_Me) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCR_red1. Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni−C bond (∼2.04 Å) in the Ni^III−methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni−C bond length suggests a homolytic cleavage of the Ni^III−methyl bond in the subsequent catalytic step. The XAS data provide insight into the role of the unique F₄₃₀ cofactor in tuning the stability of the different redox states of MCR

Fast and Selective Photoreduction of CO₂ to CO Catalyzed by a Complex of Carbon Monoxide Dehydrogenase, TiO₂, and Ag Nanoclusters

Selective, visible-light-driven conversion of CO₂ to CO with a turnover frequency of 20 s^–1 under visible light irradiation at 25 °C is catalyzed by an aqueous colloidal system comprising a pseudoternary complex formed among carbon monoxide dehydrogenase (CODH), silver nanoclusters stabilized by polymethacrylic acid (AgNCs-PMAA), and TiO₂ nanoparticles. The photocatalytic assembly, which is stable over several hours and for at least 250000 turnovers of the enzyme’s active site, was investigated by separate electrochemical (dark) and fluorescence measurements to establish specific connectivities among the components. The data show (a) that a coating of AgNCs-PMAA on TiO₂ greatly enhances its ability as an electrode for CODH-based electrocatalysis of CO₂ reduction and (b) that the individual Ag nanoclusters interact directly and dynamically with the enzyme surface, most likely at exposed cysteine thiols. The results lead to a model for photocatalysis in which the AgNCs act as photosensitizers, CODH captures the excited electrons for catalysis, and TiO₂ mediates hole transfer from the AgNC valence band to sacrificial electron donors. The results greatly increase the benchmark for reversible CO₂ reduction under ambient conditions and demonstrate that, with such efficient catalysts, the limiting factor is the supply of photogenerated electrons

X‑ray Absorption Spectroscopy Reveals an Organometallic Ni–C Bond in the CO-Treated Form of Acetyl-CoA Synthase

Acetyl-CoA synthase (ACS) is a key enzyme in the Wood–Ljungdahl pathway of anaerobic CO₂ fixation, which has long been proposed to operate by a novel mechanism involving a series of protein-bound organometallic (Ni–CO, methyl–Ni, and acetyl–Ni) intermediates. Here we report the first direct structural evidence of the proposed metal–carbon bond. We describe the preparation of the highly active metal-replete enzyme and near-quantitative generation of the kinetically competent carbonylated intermediate. This advance has allowed a combination of Ni and Fe K-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure experiments along with density functional theory calculations. The data reveal that CO binds to the proximal Ni of the six-metal metallocenter at the active site and undergoes dramatic structural and electronic perturbation in forming this organometallic Ni–CO intermediate. This direct identification of a Ni–carbon bond in the catalytically competent CO-bound form of the A cluster of ACS provides definitive experimental structural evidence supporting the proposed organometallic mechanism of anaerobic acetyl-CoA synthesis

Crystallographic Snapshots of Cyanide- and Water-Bound C-Clusters from Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase^,

Nickel-containing carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidation of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallographically characterized as either a NiFe₄S₄ or a NiFe₄S₅ metal center, the latter containing a fifth, additional sulfide that bridges Ni and a unique Fe site. To determine whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from <i>Moorella thermoacetica</i> bound both with a substrate H₂O/OH⁻ molecule and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a solution containing potassium cyanide. In both structures, the substrate H₂O/OH⁻ molecule exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H₂O/OH⁻ molecule. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidation by the C-cluster, the catalytic center of an environmentally important enzyme

Transient B₁₂-Dependent Methyltransferase Complexes Revealed by Small-Angle X‑ray Scattering

In the Wood–Ljungdahl carbon fixation pathway, protein–protein interactions between methyltransferase (MeTr) and corrinoid iron–sulfur protein (CFeSP) are required for the transfer of a methyl group. While crystal structures have been determined for MeTr and CFeSP both free and in complex, solution structures have not been established. Here, we examine the transient interactions between MeTr and CFeSP in solution using anaerobic small-angle X-ray scattering (SAXS) and present a global analysis approach for the deconvolution of heterogeneous mixtures formed by weakly interacting proteins. We further support this SAXS analysis with complementary results obtained by anaerobic isothermal titration calorimetry. Our results indicate that solution conditions affect the cooperativity with which CFeSP binds to MeTr, resulting in two distinct CFeSP/MeTr complexes with differing oligomeric compositions, both of which are active. One assembly resembles the CFeSP/MeTr complex observed crystallographically with 2:1 protein stoichiometry, while the other best fits a 1:1 CFeSP/MeTr arrangement. These results demonstrate the value of SAXS in uncovering the rich solution behavior of transient protein interactions visualized by crystallography

Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase

Protein film electrochemistry has been used to investigate reactions of highly active nickel-containing carbon monoxide dehydrogenases (CODHs). When attached to a pyrolytic graphite electrode, these enzymes behave as reversible electrocatalysts, displaying CO₂ reduction or CO oxidation at minimal overpotential. The O₂ sensitivity of CODH is suppressed by adding cyanide, a reversible inhibitor of CO oxidation, or by raising the electrode potential. Reduction of N₂O, isoelectronic with CO₂, is catalyzed by CODH, but the reaction is sluggish, despite a large overpotential, and results in inactivation. Production of H₂ and formate under highly reducing conditions is consistent with calculations predicting that a nickel-hydrido species might be formed, but the very low rates suggest that such a species is not on the main catalytic pathway

The C‑Terminal Heme Regulatory Motifs of Heme Oxygenase‑2 Are Redox-Regulated Heme Binding Sites

Heme oxygenase-2 (HO2), an enzyme that catalyzes the conversion of heme to biliverdin, contains three heme regulatory motifs (HRMs) centered at Cys127, Cys265, and Cys282. Previous studies using the soluble form of human HO2 spanning residues 1–288 (HO2_<i>sol</i>) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, heme binds to the catalytic site of HO2_<i>sol</i> via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe³⁺-heme binding upon reduction of the disulfide bond. In an effort to understand how the HRMs are involved in binding of heme to disulfide-reduced HO2_<i>sol</i>, in the work described here, we further investigated the properties of Fe³⁺-heme bound to HO2. Specifically, we investigated binding of Fe³⁺-heme to a truncated form of soluble HO2 (residues 213–288; HO2_<i>tail</i>) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2_<i>tail</i> in the disulfide-reduced state binds Fe³⁺-heme and accounts for the spectral features observed upon binding of heme to the disulfide-reduced form of HO2_<i>sol</i> that cannot be attributed to heme binding at the catalytic site. Further analysis revealed that while HO2_<i>sol</i> binds one Fe³⁺-heme per monomer of protein under oxidizing conditions, disulfide-reduced HO2_<i>sol</i> binds slightly more than two. Both Cys265 and Cys282 were identified as Fe³⁺-heme ligands, and His256 also acts as a ligand to the Cys265-ligated heme. Additionally, Fe³⁺-heme binds with a much weaker affinity to Cys282 than to Cys265, which has an affinity much weaker than that of the His45 binding site in the catalytic core. In summary, disulfide-reduced HO2 has multiple binding sites with varying affinities for Fe³⁺-heme

Spectroscopic Studies Reveal That the Heme Regulatory Motifs of Heme Oxygenase‑2 Are Dynamically Disordered and Exhibit Redox-Dependent Interaction with Heme

Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O₂- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs). While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2^O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2^R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme