Artificial Maturation Research Articles

Comparison of pore evolution for a Mesoproterozoic marine shale and a Triassic terrestrial mudstone during artificial maturation experiments

Semi-closed pyrolysis experiments were performed on a Mesoproterozoic Hongshuizhuang marine shale sample and a Triassic Chang 7 terrestrial mudstone sample, in order to understand the mechanisms of pore evolution in shales with similar organic geochemical characteristics in different depositional environments. The pore characteristics of the solid samples from the thermal maturation experiments were investigated by low-pressure gas (N2 and CO2) sorption isotherms tests and Field Emission - Scattering Electron Microscopy (FE-SEM) imaging. Results indicate the marine shale samples develop more micropores than that of the terrestrial mudstone samples during the artificial maturation. Secondary organic pores are observed in the unheated samples (~0.76% EqRo) for the marine shale, which is earlier than that of in the terrestrial mudstone (observed in the sample at 250 °C, ~0.8% Ro). Significant development of organic pores at 350 °C for both the two shales. And the transformation of smaller pores into larger pores takes place at 450 °C for the marine shale, as well as for the terrestrial mudstone. In highly overmatured shales, the overpressure formed by organic matter cracking to gas can make surrounding interparticle space to vanish. The high content of rigid minerals in the marine shale is more conducive to the preservation of organic pores than the terrestrial mudstone with rich in ductile clay minerals. Therefore, pore increases continuously for the marine shale but decreases for the terrestrial mudstone after 500 °C. Micropores would be generated if a high lithostatic pressure exists under experimental conditions, which may be largely responsible for the disagreement of pore evolution of similar shales in other publications. Finally, a conceptual model of pore evolution for type-II marine and terrestrial shales is proposed. The study reveals that the pore evolution of shales with different sedimentary environments is divergent, and this need to be separately considered in the future work.

An evaluation of kerogen molecular structures during artificial maturation

13C solid-state NMR and XPS techniques were used to investigate structural changes in kerogen during oil generation process across a range of maturity. Kerogen was isolated from an shale rock collected from Well W161 in the Dongying Depression, Bohai, China. An artificial pyrolysis experiment was carried out in a closed gold tube system using a heating rate of 2 ℃/h during which 11 temperature-point samples were collected between 350 ℃ and 450 ℃ (Easy%Ro = 0.80 to 1.98). The initial kerogen was analysed using Rock-Eval and the samples were analysed using X-ray photoelectron spectroscopy (XPS) and solid-state 13C nuclear magnetic resonance (13C NMR) to study their functional groups and chemical structures. Six averaged molecular structure models of the initial kerogen and its residues (can not be dissolved by organic reagent) were established to study the changes occurring during oil and gas generation. The results showed that with an increase in temperature, the proportion of aliphatic carbon declined while aromatic carbon increased. After reaching peak oil maturity, a condensation reaction occurred in the residue; some aromatic carbons cracked from the kerogen and combined with the residue structure. Average aromatic cluster increment was calculated to quantify the increase in the size of aromatic clusters within the structures. It was established to evaluate the changes occurring in the molecular structure of type Ⅰ kerogen during heating. Based on these results, ConU (maximum condensation degree) and ConL (lowest condensation degree) are proposed to estimate the degree of condensation of samples with different maturities.

Results of 13c nmr and ftir spectroscopy of kerogen of the upper devonian domanik of the Timan-Pechorian basin

A kerogen samples set from organic carbon rich rocks of the Middle Frasnian Early Famennian age (Domanikites) of the Timan-Pechora basin, was characterized by the Rock-Eval pyrolysis data, solid-state 13C NMR spectroscopy and Fourier transform IR spectroscopy. Additionally, the spectral characteristics of kerogen isolated from rocks artificially maturated in an autoclave in the presence of water were investigated. In general, the direction of transformation of the kerogen structure with a natural or artificial maturation coincides. For kerogen, subjected to artificial maturation, is characterized by an increase in the concentration of terminal methyl groups in relation to the methylene units. Artificial maturation of kerogen leads to a more rapid rearrangement of aromatic clusters with the accumulation of bridgehead and protonated carbon as compared with natural maturation.

Relationship between abundance and localization of maternal messenger RNA and egg quality in artificially matured Japanese eel Anguilla japonica

Artificial maturation and ovulation of Japanese eel do not always result in the production of good-quality eggs. The molecular basis for compromised egg quality in artificially matured Japanese eel remains unknown. In this study, we investigated the relationship between the localization of six maternal transcripts, grip2, dazl, sybu, trim36, pou5f3 and npm2, and egg quality in artificially matured Japanese eel oocytes and eggs. We observed that localization of these messenger RNAs (mRNAs) in oocytes from the perinucleolus stage to the migratory nucleus stage is not associated with egg quality. In contrast, in ovulated eggs, we observed two different mRNA localization patterns, which we designated type A (mRNA localized in the cortex) and type B (mRNA distributed more centrally). Furthermore, individuals containing many type B eggs tended to have low fertilization and hatching rates, but this was not reflected in the target gene mRNA levels. Our results suggest that the loss of quality in eggs from artificially matured Japanese eel is caused by abnormal mRNA localization even if there is no difference in mRNA levels.

The influence of thermal maturity on the stable isotope compositions and concentrations of molybdenum, zinc and cadmium in organic-rich marine mudrocks

The concentrations and isotopic compositions of molybdenum (Mo), zinc (Zn) and cadmium (Cd) in organic-rich marine mudrocks may be used to characterize ocean chemistry in the geological past. These approaches rely on the rarely tested assumption that the geochemical signatures of these metals are not affected by the thermal maturation of the organic matter with which they are associated. We have conducted a series of artificial maturation experiments on two well-known immature organic-rich mudrocks, the Kimmeridge Blackstone Band (Late Jurassic age), and the Posidonia Shale (Early Jurassic age). These pyrolysis experiments allow us to trace changes in the composition of organic matter through varying stages of maturation, and the concentration and isotopic compositions of metals in rock residues and evolved organic fluids. Our results indicate that the thermal maturation of organic matter does not result in significant alteration of the isotopic compositions of Mo, Zn and Cd in the rock residues, which thus retain primary palaeodepositional information. Systematic increases in the concentrations of Mo, Zn and Cd in rock residues with progressively higher thermal maturity are attributed to the loss of substrate mass in the form of fluids released during pyrolysis-induced cracking of kerogen, and to the relatively low concentrations of Mo, Zn and Cd in these fluids. The Mo-isotope compositions of fluids produced during pyrolysis are isotopically similar to the bulk rock; in contrast the isotopic composition of Zn in organic fluids is ∼0.4–0.6‰ lighter than the bulk rock. The progressive loss of organic matter from rock residues during maturation coupled with the increases in metal concentrations leads to an increase of metal/TOC ratios, which may be up to double their original (syn-depositional) value in thermally mature rocks. This observation must be taken into account when using metal/TOC ratios as proxies for oceanic metal inventories throughout geological time. Finally, calculations using the mass of asphaltenes recovered during the pyrolysis experiments suggest that organically bound Mo, Zn and Cd account for several percent of the total rock metal inventory.

Geochemical characterization and artificial thermal maturation of kerogen density fractions from the Eocene Huadian oil shale, NE China

Oil shales are an important alternative energy resource, through retorting, for the production of liquid hydrocarbons. A key factor in determining the value of an oil shale resource is its hydrocarbon generation potential; particularly its maceral distribution. To examine this, single maceral concentrates were obtained from a 1 kg Eocene Huadian oil shale sample using float-sink separation of chemically demineralized kerogen. Nine different density fractions were separated: the Botryococcus fraction occurred in the lightest (<1.06 g/mL) material, lamalginite in the 1.06–1.23 g/mL fraction, while detrovitrinite dominated in a higher density fraction (1.26–1.36 g/mL), which also contained some lamalginite.Rock-Eval S2 and hydrogen index (HI) parameters, as well as the aliphatic compound peaks in Fourier transform infrared (FTIR) spectra, all decreased with increasing kerogen density, whereas the Rock-Eval oxygen index and the aromatic compound peaks in FTIR spectra, increased. The δ13C values showed slight variations within the different density fractions. The δ13C value of parent shale kerogen was very close to that of the density fraction dominated by lamalginite (−25.1‰), because the sample is dominated by lamalginite macerals (>70%). The Botryococcus sp. fraction is isotopically enriched because Botryococcus sp. is typically significantly enriched in 13C (δ13C = −24.6‰).Artificial thermal maturation hydrocarbon generation results show that absolute hydrocarbon gas yields were similar among the different density fractions. The 1.06–1.12 g/mL fraction, dominated by lamalginite, generated nearly twice as much liquid hydrocarbons as the 1.26–1.30 g/mL fraction dominated by detrovitrinite. The maximum hydrocarbon yields corresponded well with the Rock-Eval HI values. Calculated by the relationships among HI, detrovitrinite and alginite compositions, the HI is 909 and 177 mg HC/g TOC for the purified alginite and detrovitrinite fractions, respectively. Kinetics analysis shows that Ea (activation energy) is higher for the low-density fraction (1.06–1.12 g/mL) than that for the heavy fraction (1.26–1.30 g/mL), indicating that lamalginite not only has a much better hydrocarbon generation potential, but also higher thermal stability. Therefore, this lamalginite-rich oil shale is well suited for exploitation by retorting, even though the retorting temperature would be higher than for detrovitrinite-rich fractions.

Estrogen suppresses bcl2 expression through dnmt1 in Japanese eel ovarian follicle.

Japanese eels are commercially valuable species in Asian aquaculture. This study evaluated whether salmon pituitary extract (SPE) or 17β-estradiol (E2) treatment can induce cytotoxic activity in eel ovarian follicles. Follicular cells died after exposure SPE for 24-h culture in an in vitro culture. Moreover, the E2 treatment also significantly reduced follicular cell counts. These results reveal that the inhibition of follicular cell numbers by SPE may occur through SPE-induced steroidogenesis. Results of a quantitative PCR analysis indicated that adding E2 to the culture decreased bcl2 and increased dnmt1 mRNA expression in Japanese eel follicular cells after 24 h. The results of a promoter assay revealed that E2 significantly increase dnmt1 promoter activity through estrogen receptor-binding site. An in silico analysis predicted several putative transcription factors targeting the bcl2 gene promoter region. Methylation of the bcl2 promoter accounted for the downregulation of bcl2 by E2-mediated dnmt1. The DNA methylation level of the bcl2 gene was significantly higher in E2-treated follicular cells than that in the control group. Finally, the E2-induced hypermethylation pattern of the bcl2 promoter and the reduction in follicular cell numbers were suppressed by adding an MTase inhibitor. Our findings demonstrate that estrogen has a negative effect on the reproductive system of female eels by regulating an epigenetic mechanism during artificial maturation.

Role of bitumen and NSOs during the decomposition process of a lacustrine Type-II kerogen in semi-open pyrolysis system

The purpose of this work is to investigate the generation characteristics of bitumen, NSO compounds, oil, and HC compounds during the artificial maturation of a lacustrine Type-II kerogen (which has not been given enough attention before) in order to determine its decomposition process. The analysis is based on the data itself, on the premise of jumping out of the generally accepted sequential reaction model. By taking the kerogen of the Chang 7 shale as an example, seven parallel experiments, in the temperature range from 300 °C to 420 °C were conducted on newly designed temperature-based semi-open pyrolysis system. The overall products are classified into oil and bitumen according to their phase, the C15+ fractions are classified into C15+sat, C15+aro and NSOs based on chemical compositions, and the NSOs are further classified into n-pentane NSOs and DCM NSOs according to Behar et al., (2008) and (2010). Results show that large proportion of oil is not merely a result of thermal cracking of bitumen, but also directly from the decomposition of kerogen itself. Both C15+sat and C15+aro are generated as soon as kerogen starts to decompose. It is not until the initial productivity of kerogen is basically exhausted that NSOs become the main precursor of hydrocarbons. The comparison with Behar et al. [2008, organic geochemistry 39, 1–22] further reveals that, for Chang 7 kerogen, initial decomposition of kerogen generates much more HCs than DCM NSOs. These results contradict the sequential reaction model described as: kerogen → bitumen → oil or kerogen → NSOs → hydrocarbons. Instead, they confirm the “alternate pathway” mechanism proposed by Burnham et al. [ACS symposium, 1989] in which hydrocarbons can be formed immediately from kerogen in parallel with NSOs and the formation of the two species are controlled by bond-breaking reactions that are independent of each other. This study adds geochemical insights into the decomposition mechanism of lacustrine Type-II kerogen.

Experimental Study on the Impact of Thermal Maturity on Shale Microstructures Using Hydrous Pyrolysis

Hydrous pyrolysis was applied to four low-maturity aliquots from the Utica, Excello, Monterey, and Niobrara Shale Formations in North America to create artificial maturation sequences, which could be used to study the impact of maturation on geochemical and microstructural properties. Modified Rock-Eval pyrolysis, reflectance, organic petrology, and Fourier transform infrared spectroscopy (FTIR) were employed to analyze their geochemical properties, while gas adsorption (CO2 and N2) was used to characterize their pore structures (pores < 200 nm). Organic petrography using white and blue light (fluorescence) before and after hydrous pyrolysis showed that amorphous organic matter cracked into solid bitumen, oil, and gas during hydrous pyrolysis. A reduction of the CH2/CH3 ratio in hydrous pyrolysis residues was observed from FTIR analysis. Rock-Eval pyrolysis showed that kerogens in the four samples were dissimilar, and hydrous pyrolysis residues showed smaller hydrogen index and Sh2 values than starting ma...

A preliminary experiment regarding the natural induction of gonadal development in female Japanese eels without hormone treatment

To test whether gonadal development of female eels could be promoted without any exogenous hormone treatments, we observed the effect of water temperature manipulation. After 3–5 months of water temperature treatments, three silver eels showed higher gonadosomatic indices (GSI). In particular, one eel in the 5–15°C fluctuating temperature treatment group (5°C daytime and 15°C night‐time) had the highest GSI of 8.5 with secondary yolk globule stage oocytes of large diameter (OD; 412 μm) after 3 months, which indicated definite gonadal development compared with those in the initial states (mean GSI, 2.4; OD, 226.7 μm). The 5–15°C fluctuating temperature and constant 5°C groups had low oocyte breakdown (atretic) rates. Because daily temperature fluctuations and cold daytime water are experienced by vertically migrating silver eels in the ocean, these temperature conditions may be key maturation process components that could be useful for hormone‐free artificial maturation.

Understanding organic matter heterogeneity and maturation rate by Raman spectroscopy

Solid organic matter (OM) in sedimentary rocks produces petroleum and solid bitumen when it undergoes thermal maturation. The solid OM is a ‘geomacromolecule’, usually representing a mixture of various organisms with distinct biogenic origins, and can have high heterogeneity in composition. Programmed pyrolysis is a common method to reveal bulk geochemical characteristics of the dominant OM, while detailed organic petrography is required to reveal information about the biogenic origin of contributing macerals. Despite the advantages of programmed pyrolysis, it cannot provide information about the heterogeneity of chemical compositions present in the individual OM types. Therefore, other analytical techniques such as Raman spectroscopy are necessary.In this study, we compared geochemical characteristics and Raman spectra of two sets of naturally and artificially matured Bakken source rock samples. A continuous Raman spectral map on solid bitumen particles was created from the artificially matured hydrous pyrolysis residues, in particular, to show the systematic chemical modifications in microscale. Spectroscopic data was plotted for both sets against thermal maturity to compare maturation rate/path for these two separate groups. The outcome showed that artificial maturation through hydrous pyrolysis does not follow the same trend as naturally-matured samples although having similar solid bitumen reflectance values (%SBRo).Furthermore, Raman spectroscopy of solid bitumen from artificially matured samples indicated the heterogeneity of OM decreases as maturity increases. This may represent an alteration in chemical structure towards more uniform compounds at higher maturity. This study may emphasize the necessity of using analytical methods such as Raman spectroscopy along with conventional geochemical methods to better reveal the underlying chemical structure of OM. Finally, observation by Raman spectroscopy of chemical alteration of OM during artificial maturation may assist in the proposal of improved pyrolysis protocols to better resemble natural geologic processes.

The material balance of organic matter of Domanic shale formation after thermal treatment

In this paper, we discuss the amount of generated gaseous and liquid hydrocarbons as a product of artificial maturation of organic matters of Domanic black shale. The material balance of organic matter for initial rock sample and after thermal treatment at 300 and 500 °С were estimated. The amount of generated liquid hydrocarbon was minimum at 500 °С. As a result of kerogen destruction, no asphaltenes were observed during generation of liquid hydrocarbons. Based on the results of elemental analysis, Van Krevelen diagram was plotted.

Synthesis of a miniaturized [FeFe] hydrogenase model system.

The reaction occurring during artificial maturation of [FeFe] hydrogenase has been recreated using molecular systems. The formation of a miniaturized [FeFe] hydrogenase model system, generated through the combination of a [4Fe4S] cluster binding oligopeptide and an organometallic Fe complex, has been monitored by a range of spectroscopic techniques. A structure of the final assembly is suggested based on EPR and FTIR spectroscopy in combination with DFT calculations. The capacity of this novel H-cluster model to catalyze H2 production in aqueous media at mild potentials is verified in chemical assays.

Evolution characteristics and model of nanopore structure and adsorption capacity in organic-rich shale during artificial thermal maturation: A pyrolysis study of the Mesoproterozoic Xiamaling marine shale with type II kerogen from Zhangjiakou, Hebei, China

Thermal maturity has a considerable impact on hydrocarbon generation, mineral conversion, nanopore structure, and adsorption capacity evolution of shale, but that impact on organic-rich marine shales containing type II kerogen has been rarely subjected to explicit and quantitative characterization. This study aims to obtain information regarding the effects of thermal maturation on organic matter, mineral content, pore structure, and adsorption capacity evolution of marine shale. Mesoproterozoic Xiamaling immaturity marine oil shale with type II kerogen in Zhangjiakou of Hebei, China, was chosen for anhydrous pyrolysis to simulate the maturation process. With increasing simulation temperature, hydrocarbon generation and mineral transformation promote the formation, development, and evolution of pores in the shale. The original and simulated samples consist of closed microspores and one-end closed pores of the slit throat, all-opened wedge-shaped capillaries, and fractured or lamellar pores, which are related to the plate particles of clay. The increase in maturity can promote the formation and development of pores in the shale. Heating can also promote the accumulation, formation, and development of pores, leading to a large pore volume and surface area. The temperature increase can promote the development of pore volume and surface area of 1–10 and 40-nm diameter pores. The formation and development of pore volume and surface area of 1–10 nm diameter pores are more substantial than that of 40-nm diameter pores. The pore structure evolution of the sample can be divided into pore adjustment (T &lt; 350°C, EqRo &lt; 0.86%), development (350°C &lt; T &lt; 650°C, 0.86% &lt; EqRo &lt; 3.28%), and conversion or destruction stages (T &gt; 650°C, EqRo &gt; 3.28%). Along with the increase in maturity, the methane adsorption content decreases in the initial simulation stage, increases in the middle simulation stage, and reaches the maximum value at 650°C, after which it gradually decreases. A general evolution model is proposed by combining the nanopore structure and the adsorption capacity evolution characteristics of the oil shale.

Application of Raman Spectroscopy as Thermal Maturity Probe in Shale Petroleum Systems: Insights from Natural and Artificial Maturation Series

Raman spectroscopy was studied as a thermal maturity probe in a series of Upper Devonian Ohio Shale samples from the Appalachian Basin spanning from immature to dry gas conditions. Raman spectroscopy also was applied to samples spanning a similar thermal range created from 72-h hydrous pyrolysis (HP) experiments of the Ohio Shale at temperatures from 300 to 360 °C and isothermal HP experiments lasting up to 100 days of similar Devonian–Mississippian New Albany Shale. Raman spectra were treated by automated evaluation software based on iterative and simultaneous modeling of signal and baseline functions to decrease subjectivity. Spectra show robust correlation to measured solid bitumen reflectance (BRo) values and were therefore used to construct logarithmic regression relationships for calculation of BRo equivalent values. Raman spectra show considerable differences between natural samples and HP residues with similar measured BRo values, indicating as-yet undetermined differences in carbon chemistry. We ...

Organic petrographic analysis of artificially matured chitinozoan- and graptolite-rich Upper Ordovician shale from Hudson Bay Basin, Canada

Chitinozoan, graptolite and organic-rich immature Upper Ordovician mudstone was artificially matured using closed hydrous pyrolysis. The pyrolysis was performed at isothermal temperatures of 310-350 °C at 10 °C increments for 72 h. This temperature range simulates the subsurface thermogenic hydrocarbon generation window based on previous laboratory simulations. The objectives of this study are to (a) qualitatively and quantitatively assess the degree of physicochemical transformation of dispersed organic matter (DOM) and zooclasts in BRF petroleum source rock after artificial maturation; and (b) determine the relationship between graptolite, chitinozoan, vitrinite-like particle and bitumen reflectance (Ro) with increasing pyrolysis temperature and the thermal maturity. Petrographic analysis shows that bituminite and amorphous kerogen were the first organic macerals to thermally decompose after the first pyrolysis temperature. This is followed by liptodetrinite, thin-walled alginite, acritarch, and telalginite as pyrolysis temperature increases. Concurrently, the fluorescence properties of the telalginite shifted from greenish yellow to reddish-orange after the last stage (350 °C) pyrolysis. In addition, significant amount of bright fluorescing labile hydrocarbons produced during pyrolysis were observed oozing from pore spaces and on the surface of the rock matrix. Pore-filling, orange to reddish-orange fluorescing solid bitumen were also found in pores spaces created by the thermal decomposition of DOM. The lack of morphological evidence for the thermal decomposition of chitinozoans and graptolites suggests that these zooclasts macerals may have limited or have no contribution to the overall hydrocarbon generation. Nonetheless, their respective reflectance (Ro) increased (0.64% to 1.34% and 0.55% to 1.38%) as the pyrolysis temperature was increased. These Ro values are significantly higher than the vitrinite-like particles (0.49% to 1.07%) and solid bitumen (0.30% to 1.09%). The measured RocK-Eval Tmax and solid bitumen vitrinite reflectance equivalent (VRo-eqv) are comparable to the average vitrinite-like particles. The relationship between rate of increase in chitinozoan and graptolite, and vitrinite-like particles Ro, expressed by the equations: Ro-vit-like = 0.77 Rchi and Ro-vit-like = 0.79 Rgrap, are comparable to those observed in geologically matured source rocks.

Nanoscale Pore Changes in a Marine Shale: A Case Study Using Pyrolysis Experiments and Nitrogen Adsorption

Nanoscale pores have an important role in the accumulation of gas in shale gas reservoirs. Indeed, the formation of nanopores is critical for the characterization and evaluation of a shale reservoir. Moreover, the effect of pyrolysis on the modification of nanopores is not clear. Therefore, this paper focuses on pyrolysis and nitrogen adsorption experiments to examine the nanoscale pore structure and evolution in marine shale strata with low total organic carbon content. All of the examined samples contain micro-, meso-, and macropores. The results show that the number of micropores increased as a result of artificial maturation (i.e., pyrolysis), which resulted in a significant increase in the surface area and the total pore volume. The openness of the pores significantly increased when the maturity was higher than 2.5% Ro (vitrinite reflectance). The 1.5–7.5 and 60–70 nm pores are the most pronounced to change after pyrolysis. Furthermore, liquid hydrocarbons produced during heating were shown to influe...

Cyanide Docking and Linkage Isomerism in Models for the Artificial [FeFe]-Hydrogenase Maturation Process.

Linkage isomerization of the cyanide on the [2Fe] subsite of the [FeFe]-H2ase active site was reported to occur during the docking of various synthetic diiron complexes onto a carrier protein, apo-HydF, as the initial step for the artificial maturation of the [FeFe]-H2ase enzyme (Berggren et al., Nature, 2013, 499, 66-70). An investigation of our triiron organometallic models (FeFe-CN/NC-Fe') revealed that, once a Fe-CN-Fe connection is formed, high barriers prevent such cyanide linkage isomerization ( Chem. Sci., 2016, 7, 3710-3719). To explore effects of variable oxidation states of the receiver unit, we introduce copper(I/II) fragments, precedented in Holm's models of cytochrome c oxidase to induce cyanide isomerization (Cu-CN/NC-Fe), to the diiron synthetic analogues of [FeFe]-H2ase. For comparison, a zinc variant of the cytochrome c oxidase model is also examined. According to the oxidation state of copper, a cyanide flip was induced during the formation of both Zn-NC-Cu and FeFe-CN-Cu complexes. Density functional theory calculations are used to predict the mechanisms for such linkage isomerization and account for optimal conditions including oxidation states of metals, spin states, and solvation. These results on synthetic paradigms imply a role for oxidation state control of cyanide isomerization during hydrogenase active site assembly.

Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants

[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2−, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2−. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2−, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe–4S] subcluster. The reduced form of HydA1 containing only the [4Fe–4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]–PDT/ADT complex.

A [RuRu] Analogue of an [FeFe]-Hydrogenase Traps the Key Hydride Intermediate of the Catalytic Cycle.

The active site of the [FeFe]-hydrogenases features a binuclear [2Fe]H sub-cluster that contains a unique bridging amine moiety close to an exposed iron center. Heterolytic splitting of H2 results in the formation of a transient terminal hydride at this iron site, which, however is difficult to stabilize. We show that the hydride intermediate forms immediately when [2Fe]H is replaced with [2Ru]H analogues through artificial maturation. Outside the protein, the [2Ru]H analogues form bridging hydrides, which rearrange to terminal hydrides after insertion into the apo-protein. H/D exchange of the hydride only occurs for [2Ru]H analogues containing the bridging amine moiety.