In situ electrochemical regeneration of active 1,4-NADH for enzymatic lactic acid formation via concerted functions on Pt-modified TiO2/Ti.

Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are key cofactors serving as essential hydrogen acceptors and donors to facilitate energy and material conversions under mild conditions. We demonstrate direct electrochemical conversion to achieve highly efficient regeneration of enzymatically active 1,4-NADH using a Pt-modified TiO2 catalyst grown directly on a Ti mesh electrode (Pt-TOT). Spectral analyses revealed that defects formed by the inclusion of Pt species in the lattice of TiO2 play a critical role in the regeneration process. In particular, Pt-TOT containing approximately 3 atom% of Pt exhibited unprecedented efficiency in the electrochemical reduction of NAD+ at the lowest overpotential to date. This exceptional performance led to the production of active 1,4-NADH with a significantly high yield of 86 ± 3% at -0.6 V vs. Ag/AgCl (-0.06 V vs. RHE) and an even higher yield of 99.5 ± 0.4% at a slightly elevated negative potential of -0.8 V vs. Ag/AgCl (-0.2 V vs. RHE). Furthermore, the electrochemically generated NADH was directly applied in the enzymatic conversion of pyruvic acid to lactic acid using lactate dehydrogenase.

Disassembly and reassembly of the non-conventional thermophilic C-phycocyanin.

C-phycocyanin (CPC), which contains open-chain tetrapyrroles, is a major light-harvesting red-fluorescent protein with an important role in aquatic photosynthesis. Recently, we reported a non-conventional CPC from Thermoleptolyngbya sp. O-77 (CPCO77) that contains two different structures, i.e., a hexameric structure and a non-conventional octameric structure. However, the assembly and disassembly mechanisms of the non-conventional octameric form of CPC remain unclear. To understand this assembly mechanism, we performed an in vitro experiment to study the disassembly and reassembly behaviors of CPC using isolated CPC subunits. The dissociation of the CPCO77 subunit was performed using a Phenyl-Sepharose column in 20 mM potassium phosphate buffer (pH 6.0) containing 7.0 M urea. For the first time, crystals of isolated CPC subunits were obtained and analyzed after separation. After the removal of urea from the purified α and ß subunits, we performed an in vitro reassembly experiment for CPC and analyzed the reconstructed CPC using spectrophotometric and X-ray crystal structure analyses. The crystal structure of the reassembled CPC was nearly identical to that of the original CPCO77. The findings of this study indicate that the octameric CPCO77 is a naturally occurring form in the thermophilic cyanobacterium Thermoleptolyngbya sp. O-77.

Storing electrons from H2 for transfer to CO2, all at room temperature.

We present an Ir complex that extracts electrons from H2 at room temperature and stores them as a H2-derived energy carrier (H2EC) at room temperature. Furthermore, we demonstrate that this complex reduces CO2 to a metal-CO22- species at room temperature, and present the first electrospray ionisation mass spectrum for this compound.

H2-driven reduction of CO2 to formate using bacterial plasma membranes.

Bacterial membranes shield the intracellular compartment by selectively allowing unwanted substances to enter in, which in turn reduces overall catalytic efficiency. This report presents a model system using the isolated plasma membranes of Citrobacter sp. S-77 that harbor oxygen-stable [NiFe]hydrogenase and [Mo]formate dehydrogenase, which are integrated into a natural catalytic nanodevice through an electron transfer relay. This naturally occurring nanodevice exhibited selectivity and efficiency in catalyzing the H2-driven conversion of CO2 to formate with the rate of 817 mmol·L-1·gprotein-1·h-1 under mild conditions of 30 °C, pH 7.0, and 0.1 MPa. When the isolated plasma membranes of Citrobacter sp. S-77 was immobilized with multi-walled carbon nanotubes and encapsulated in hydrogel beads of gellan-gum cross-linked with calcium ions, the catalyst for formate production remained stable over 10 repeated uses. This paper reports the first case of efficient and selective formate production from H2 and CO2 using bacterial plasma membranes.

Selective formate production from H2 and CO2 using encapsulated whole-cells under mild reaction conditions.

Biocatalytic CO2 reduction into formate is a crucial strategy for developing clean energy because formate is considered as one of the promising hydrogen storage materials for achieving net-zero carbon emissions. Here, we developed an efficient biocatalytic system to produce formate selectively by coupling two enzymatic activities of H2 oxidation and CO2 reduction using encapsulated bacterial cells of Citrobacter sp. S-77. The encapsulated whole-cell catalyst was made by living cells depositing into polyvinyl alcohol and gellan gum cross-linked by calcium ions to form hydrogel beads. Formate production using encapsulated cells was carried out under the resting state conditions in the gas mixture of H2/CO2 (70:30, v/v%). The whole-cell biocatalyst showed highly efficient and selective catalytic production of formate, reaching the specific rate of formate production of 110 mmol L-1· gprotein-1·h-1 at 30 °C, pH 7.0, and 0.1 MPa. The encapsulated cells can be reused at least 8 times while keeping their high catalytic activities for formate production under mild reaction conditions.

C-H Arylation of Benzene with Aryl Halides using H2 and a Water-Soluble Rh-Based Electron Storage Catalyst.

This paper reports the first example of C-H arylation of benzene under mild conditions, using H2 as an electron source {turnover numbers (TONs)=0.7-2.0 for 24 h}. The reaction depends on a Rh-based electron storage catalyst, and proceeds at room temperature and in aqueous solution. Furthermore, the H2 is inactive during the radical transfer step, greatly reducing unwanted side reactions.

Non-conventional octameric structure of C-phycocyanin.

C-phycocyanin (CPC), a blue pigment protein, is an indispensable component of giant phycobilisomes, which are light-harvesting antenna complexes in cyanobacteria that transfer energy efficiently to photosystems I and II. X-ray crystallographic and electron microscopy (EM) analyses have revealed the structure of CPC to be a closed toroidal hexamer by assembling two trimers. In this study, the structural characterization of non-conventional octameric CPC is reported for the first time. Analyses of the crystal and cryogenic EM structures of the native CPC from filamentous thermophilic cyanobacterium Thermoleptolyngbya sp. O-77 unexpectedly illustrated the coexistence of conventional hexamer and novel octamer. In addition, an unusual dimeric state, observed via analytical ultracentrifugation, was postulated to be a key intermediate structure in the assemble of the previously unobserved octamer. These observations provide new insights into the assembly processes of CPCs and the mechanism of energy transfer in the light-harvesting complexes.

Synthesis of acetic acid from CO2, CH3I and H2 using a water-soluble electron storage catalyst.

This paper reports a possible mechanism of acetic acid formation from CO2, CH3I and H2 in aqueous media and the central role played by a water-soluble Rh-based electron storage catalyst. In addition to water-solubility, we also report the crystal structures of two presumed intermediates. These findings together reveal (1) the advantage of water, not only as a green solvent, but also as a reactive Lewis base to extract H+ from H2, (2) the role of the metal (Rh) centre as a point for storing electrons from H2 and (3) the importance of an electron-withdrawing ligand (quaterpyridine, qpy) that supports electron storage.

Reductive C(sp3)-C(sp3) homo-coupling of benzyl or allyl halides with H2 using a water-soluble electron storage catalyst.

This paper reports the first example of a reductive C(sp3)-C(sp3) homo-coupling of benzyl/allyl halides in aqueous solution by using H2 as an electron source {turnover numbers (TONs) = 0.5-2.3 for 12 h}. This homo-coupling reaction, promoted by visible light, is catalysed by a water-soluble electron storage catalyst (ESC). The reaction mechanism, and four requirements to make it possible, are also described.

A NiRhS fuel cell catalyst - lessons from hydrogenase.

We present a novel fuel cell heterogeneous catalyst based on rhodium, nickel and sulfur with power densities 5-28% that of platinum. The NiRhS heterogeneous catalyst was developed via a homogeneous model complex of the [NiFe]hydrogenases (H2ases) and can act as both the cathode and anode of a fuel cell.

[NiFe], [FeFe], and [Fe] hydrogenase models from isomers.

The study of hydrogenase enzymes (H2ases) is necessary because of their importance to a future hydrogen energy economy. These enzymes come in three distinct classes: [NiFe] H2ases, which have a propensity toward H2 oxidation; [FeFe] H2ases, which have a propensity toward H2 evolution; and [Fe] H2ases, which catalyze H- transfer. Modeling these enzymes has so far treated them as different species, which is understandable given the different cores and ligand sets of the natural molecules. Here, we demonstrate, using x-ray analysis and nuclear magnetic resonance, infrared, Mössbauer spectroscopies, and electrochemical measurement, that the catalytic properties of all three enzymes can be mimicked with only three isomers of the same NiFe complex.

Biochemical and structural characterization of a thermostable Dps protein with His-type ferroxidase centers and outer metal-binding sites.

The DNA-binding protein from starved cells (Dps) is found in a wide range of microorganisms, and it has been well characterized. However, little is known about Dps proteins from nonheterocystous filamentous cyanobacteria. In this study, a Dps protein from the thermophilic nonheterocystous filamentous cyanobacterium Thermoleptolyngbya sp. O-77 (TlDps1) was purified and characterized. PAGE and CD analyses of TlDps1 demonstrated that it had higher thermostability than previously reported Dps proteins. X-ray crystallographic analysis revealed that TlDps1 possessed His-type ferroxidase centers within the cavity and unique metal-binding sites located on the surface of the protein, which presumably contributed to its exceedingly high thermostability.

Glyceraldehyde-3-phosphate dehydrogenase from Citrobacter sp. S-77 is post-translationally modified by CoA (protein CoAlation) under oxidative stress.

Protein CoAlation (S-thiolation by coenzyme A) has recently emerged as an alternative redox-regulated post-translational modification by which protein thiols are covalently modified with coenzyme A (CoA). However, little is known about the role and mechanism of this post-translational modification. In the present study, we investigated CoAlation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a facultative anaerobic Gram-negative bacterium Citrobacter sp. S-77 (Cb GAPDH). GAPDH is a key glycolytic enzyme whose activity relies on the thiol-based redox-regulated post-translational modifications of active-site cysteine. LC-MS/MS analysis revealed that CoAlation of Cb GAPDH occurred in vivo under sodium hypochlorite (NaOCl) stress. The purified Cb GAPDH was highly sensitive to overoxidation by H2O2 and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H2O2/NaOCl in the presence of CoA led to CoAlation and inactivation of the enzyme; activity could be recovered after incubation with dithiothreitol, glutathione and CoA. CoAlation of the enzyme in vitro was confirmed by mass spectrometry. The presence of a substrate, glyceraldehyde-3-phosphate, fully protected Cb GAPDH from inactivation by CoAlation, suggesting that the inactivation is due to the formation of mixed disulphides between CoA and the active-site cysteine Cys149. A molecular docking study also supported the formation of mixed disulphide without steric constraints. These observations suggest that CoAlation is an alternative mechanism to protect the redox-sensitive thiol (Cys149) of Cb GAPDH against irreversible oxidation, thereby regulating enzyme activity under oxidative stress.

Redox-dependent conformational changes of a proximal [4Fe-4S] cluster in Hyb-type [NiFe]-hydrogenase to protect the active site from O2.

Citrobacter sp. S-77 [NiFe]-hydrogenase harbors a standard [4Fe-4S] cluster proximal to the Ni-Fe active site. The presence of relocatable water molecules and a flexible aspartate enables the [4Fe-4S] to display redox-dependent conformational changes. These structural features are proposed to be the key aspects that protect the active site from O2 attack.

Multifunctional Catalysts for H2 O2 -Resistant Hydrogen Fuel Cells.

The development of hydrogen fuel cells is greatly hindered by the unwanted generation of H2 O2 at the cathode. A non-Pt cathode catalyst is now shown to be capable of simultaneously reducing both O2 and H2 O2 , thus rendering H2 O2 a useful part of the feed stream. The applicability of this unique catalyst is demonstrated by employing it in a fuel cell running on H2 /CO and O2 /H2 O2 .

Oxidation of Guanosine Monophosphate with O2 via a Ru-peroxo Complex in Water.

Oxidative damage of DNA by reactive oxygen species (ROS) is responsible for aging and cancer. Although many studies of DNA damage by ROS have been conducted, there have been no reports of the oxidation of RNA components, such as guanosine monophosphate, by metal-based species in water. Here, we report the first case of oxidation of guanosine monophosphate to 8-oxoguanosine monophosphate by a metal-based oxygen bound species, derived from O2 and in water.

Mechanistic investigation of the formation of H2 from HCOOH with a dinuclear Ru model complex for formate hydrogen lyase.

We report the mechanistic investigation of catalytic H2 evolution from formic acid in water using a formate-bridged dinuclear Ru complex as a formate hydrogen lyase model. The mechanistic study is based on isotope-labeling experiments involving hydrogen isotope exchange reaction.

Nitrogen Fixation Genes and Nitrogenase Activity of the Non-Heterocystous Cyanobacterium Thermoleptolyngbya sp. O-77.

Cyanobacteria are widely distributed in marine, aquatic, and terrestrial ecosystems, and play an important role in the global nitrogen cycle. In the present study, we examined the genome sequence of the thermophilic non-heterocystous N2-fixing cyanobacterium, Thermoleptolyngbya sp. O-77 (formerly known as Leptolyngbya sp. O-77) and characterized its nitrogenase activity. The genome of this cyanobacterial strain O-77 consists of a single chromosome containing a nitrogen fixation gene cluster. A phylogenetic analysis indicated that the NifH amino acid sequence from strain O-77 was clustered with those from a group of mesophilic species: the highest identity was found in Leptolyngbya sp. KIOST-1 (97.9% sequence identity). The nitrogenase activity of O-77 cells was dependent on illumination, whereas a high intensity of light of 40 µmol m-2 s-1 suppressed the effects of illumination.

One Model, Two Enzymes: Activation of Hydrogen and Carbon Monoxide.

The ability to catalyze the oxidation of both H2 and CO in one reaction pot would be a major boon to hydrogen technology since CO is a consistent contaminant of H2 supplies. Here, we report just such a catalyst, with the ability to catalyze the oxidation of either or both H2 and CO, based on the pH value. This catalyst is based on a NiIr core that mimics the chemical function of [NiFe]hydrogenase in acidic media (pH 4-7) and carbon monoxide dehydrogenase in basic media (pH 7-10). We have applied this catalyst in a demonstration fuel cell using H2 , CO, and H2 /CO (1/1) feeds as fuels for oxidation at the anode. The power density of the fuel cell depends on the pH value in the media of the fuel cell and shows a similar pH dependence in a flask. We have isolated and characterized all intermediates in our proposed catalytic cycles.

Acetyl-CoA production by encapsulated pyruvate ferredoxin oxidoreductase in alginate hydrogels.

Pyruvate ferredoxin oxidoreductase from Citrobacter sp. S-77 (PFORS77) was purified in order to develop a method for acetyl-CoA production. Although the purified PFORS77 showed high O2-sensitivity, the activity could be remarkably stabilized in anaerobic conditions. PFORS77 was effectively immobilized on ceramic hydroxyapatite (PFORS77-HA) with an efficiency of more than 96%, however, after encapsulation of PFORS77-HA in alginate, the rate of catalytic acetyl-CoA production was highly reduced to 36% when compared to that of the free enzyme. However, the operational stability of the PFORS77-HA in alginate hydrogels was remarkable, retaining over 68% initial activity even after ten repeated cycles. The results suggested that the PFORS77-HA hydrogels have a high potential for biotechnological application.