Synthetic photobiocatalysts are promising catalysts for valuable chemical transformations by harnessing solar energy inspired by natural photosynthesis. However, the synergistic integration of all of the components for efficient light harvesting, cascade electron transfer, and efficient biocatalytic reactions presents a formidable challenge. In particular, replicating intricate multiscale hierarchical assembly and functional segregation involved in natural photosystems, such as photosystems I and II, remains particularly demanding within artificial structures. Here, we report the bottom-up construction of a visible-light-driven chemical-biological hybrid nanoreactor with augmented photocatalytic efficiency by anchoring an α-carboxysome shell encasing [FeFe]-hydrogenases (H-S) on the surface of a hydrogen-bonded organic molecular crystal, a microporous α-polymorph of 1,3,6,8-tetra(4'-carboxyphenyl)pyrene (TBAP-α). The self-association of this chemical-biological hybrid system is facilitated by hydrogen bonds, as revealed by molecular dynamics simulations. Within this hybrid photobiocatalyst, TBAP-α functions as an antenna for visible-light absorption and exciton generation, supplying electrons for sacrificial hydrogen production by H-S in aqueous solutions. This coordination allows the hybrid nanoreactor, H-S|TBAP-α, to execute hydrogen evolution exclusively driven by light irradiation with a rate comparable to that of photocatalyst-loaded precious cocatalyst. The established approach to constructing new light-driven biocatalysts combines the synergistic power of biological nanotechnology with the multilength-scale structure and functional control offered by supramolecular organic semiconductors. It opens up innovative opportunities for the fabrication of biomimetic nanoreactors for sustainable fuel production and enzymatic reactions.

SUMMARYMicroalgae have been positioned as excellent models for producing new sources of energy (biofuels and biohydrogen). Some investigations in these biological models have been directed to know if the enzymes ferredoxin (FDX) and hydrogenase (HYD) are involved in the algae producing different concentrations of molecular hydrogen (H2). To date, little is known about the concomitant transcriptional regulation of both enzymes during H2 evolution in algae. In this research, we evaluated the relative expression of hdy and fdx genes during the evolution of H2 in three microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii) in N‐deprived anaerobic cultures in the presence of Fe, and 12:24 and 24:24 h dark:light cycles. We also detected structural differences in the enzymes. The 3D modeling indicated that the 3D structure of HYD and FDX are conserved in most algal genera, and the results of our grouping according to the aa characteristics of the proteins showed two grouping trends: One, according to the algae's phylogenetic classification, and another one according to the species‐specific enzyme's characteristics, and the grouping could perhaps be more influenced by the algae's ability to produce H2. The three microalgae species reached maximum H2 accumulation values in 24h:24 h dark:light conditions in Fe‐supplemented media (4.2 ± 0.12 mL L−1 in C. vulgaris, 3.9 ± 0.10 mL L−1 in S. obliquus, and 4.5 ± 0.10 mL L−1 in C. reinhardtii), and the highest global relative expression of hyd and fdx genes was reached during the first hour of exposure to light, which suggests concomitant expression of both enzymes at the beginning of H2 production. The behavior of the expression of the hyd and fdx genes in these algal species proved to be similar between species. A better understanding of the concomitant regulation of both enzymes could lay the groundwork for the future use of both enzymes to improve H2 yields in microalgae.

[Fe‐Fe] hydrogenases are enzymes that perform a reversible heterolytic splitting of hydrogen into two protons and two electrons. The major roadblock in the utilization of these very efficient catalysts in renewable energy devices is the irreversible damage by oxygen and reactive oxygen species. The exact mechanism of the oxygen‐triggered inhibition is not known. However, it has been shown that in the presence of oxygen the active 6Fe metallocenter (H‐cluster) is irreversibly degraded, perhaps via a generation of a reactive oxygen species that react with components of the H‐cluster. In this work, we illustrate that an [Fe‐Fe] hydrogenase from Clostridium beijerinckii shows staggering tolerance to oxygen in atmospheric concentrations. Using activity assays and Fourier Transform infrared (FTIR) spectrometry we illustrate that the H‐cluster forms a stable adduct with oxygen that can be readily reactivated in the presence of hydrogen. Using protein film voltametry (PFV), IR spectroelectromemistry and EPR spectroscopy we provide a detailed description of the oxygen‐reacted state of the active center. Guided by bioinformatics analysis of the [Fe‐Fe] hydrogense family, we performed amino acid substitutions and subsequent biophysical characterization of the variants. The results of this study allow us to delineate the role of protein surrounding of the H‐cluster and of the auxiliary [4Fe‐4S] clusters in facilitating oxygen tolerance and catalytic bias observed in our PFV experiments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correction to "Sulfide Protects [FeFe] Hydrogenases From O2".

Air-stable [FeFe] hydrogenases

Interaction of Eu(fod)3 with coordinated cyanide in an analogue of the sub-site of [FeFe]-hydrogenase

The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.Electronic supplementary materialThe online version of this article (doi:10.1007/s11120-015-0198-6) contains supplementary material, which is available to authorized users.

Objective To study the expression of GRHL-3 in diffuse large B-cell lymphoma (DLBCL) tissues and its clinical significance. Methods One hundred and sixty-eight pathology paraffin-embedded diffuse large B-cell lymphomas tissues were collected from January 2006 to September 2011. Immunohistochemistry was used to detect the expression of GRHL-3 protein in the above tissues. Results The positive expression rate of GRHL-3 protein in the GCB type tissues was higher than that in the non-GCB type tissues [84.87 %(101/119) vs 14.29 % (7/49), P < 0.01]. Further analysis indicated that in the non-GCB type tissues,the positive expression rate of GRHL-3 in the latter stage group was significantly higher than that in the early stage group [90.00 % (63/70) vs 77.56 % (38/49), P < 0.01]. The positive expression rate of GRHL-3 in the lactatede hydrogenase increased group was significantly higher than that in the normal lactated hydrogenase [91.67 % (77/84) vs 68.57 % (24/35), P < 0.01]. The positive expression rate of GRHL-3 in the extranodal involvement status ≥2 group was significantly higher than that in the extranodal involvement status 0-1 group [96.29 % (26/27) vs 81.52 % (75/92), P < 0.05]. The positive expression rate of GRHL-3 in the IPI score 4-5 group was significantly higher than that in the IPI score 0-1 group [91.30 % (65/69) vs 66.67 % (18/27), P < 0.01] and IPI score 2-3 group [91.30 % (65/69) vs 79.96 % (18/23), P < 0.05]. However, the expression of GRHL-3 had no correlation with the gender, age, and performance status of DLBCL. Conclusion The positive expression rate of GRHL-3 protein in the GCB type tissues is higher than that in the non-GCB type tissues. The positive expression rate of GRHL-3 in the DLBCL is correlated with the Ann Arbor stage, lactate dehydrogenase, extranodal involvement status and IPI score. Key words: Lymphoma, large B-cell, diffuse; GRHL-3; Immunohistochemistry

Hydrogenase enzymes,which catalyze the formation and dissociation of hydrogen are heteromeric metalloenzymes. Mature hydrogenases are usually highly sensitive to oxygen,and the pro-enzymes are not active unless they are modified by a complicated post-transltational maturation process which involves synergized work on catalytic center of the enzymes by related chaprons. Catalytic mechanisms of hydrogenases also plays pivotal role in development of valuable oxygen-resistant biocatalysts for bio-hydrogen production and synthetic hydrogenase mimics applied in green battery industry. Recombinant enzymes are therefore indispensable for enzymes 'structural studies,since acquisition of native enzymes is extremely difficult,and,in some case impossible. This review aims to summarize and analyze recent progress in studies using native or foreign hosts to achive successful expression of recombinant enzymes that are either iron only or Ni Fe containing. Furthermore,the enzymatic features were systematically compared between native and recombinant proteins,and the likely solutions for future works in this area were also proposed.

Hydrogenases

ABSTRACT A brief discussion is presented of recent information concerning (a) factors influencing extent of N2, loss during N2 fixation by legumes; (b) electron carriers involved in the oxyhydrogen reaction of Rhizobium japonicum bacteroids; and (c) progress made in evaluating H2 recycling advantages. A major factor determining whether H2 is evolved from legume nodules is the presence of an active uptake hydrogenase which participates in the oxidation of H2 that is evolved as a by-product of the nitrogenase reaction. The extent of H2 evolution from the nitrogenase reaction is affected by those factors that influence the nitrogenase turnover rate. These include the supply of ATP and reductant and the ratio of the Fe protein to the MoFe protein component of nitrogenase. Oxidation of H2 in Rhizobium bacteroids is catalyzed by a series of enzymes located in bacteroid membranes. In addition to the hydrogenase per se, carriers so far shown to be involved in the process include cytochromes of the b and c types a...

合成了以三联吡啶锇Os（Ⅱ）配合物为光敏剂的PS-Fe2S2型模拟铁氢化酶分子光催化剂1a及其分子间光催化模型化合物1b和2,研究了配合物1a和1b的吸收光谱,发光光谱及电化学性质.配合物1a和1b均表现出三联吡啶锇Os（Ⅱ）配合物的MLCT吸收峰;与不含Fe2S2基团的配合物1b相比,在配合物1a中三联吡啶锇Os（Ⅱ）配合物单元的发光被明显猝灭,猝灭程度为92%.而在同样浓度下,配合物1b与2组成的分子间体系中三联吡啶锇Os（Ⅱ）配合物的发光仅被猝灭了4%.通过Rehm-Weller方程计算得出由三联吡啶锇Os（Ⅱ）配合物单元到Fe2S2活性中心的光致电子转移自由能为正,表明分子内1a和分子间1b＋2体系均不能发生光致电子转移,体系发光猝灭的原因是三联吡啶锇Os（Ⅱ）配合物3MLCT激发态与铁氢化酶模拟活性中心Fe2S2的能量转移.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

H2 is an ideal, clean and potentially sustainable energy carrier for the future due to its large energy content per weight, abundance and non-polluting nature. The selection of optimal H2 production technology depends on the H2-producing enzymes available. Thiocapsa roseopersicina contains a nitrogenase and several [NiFe] hydrogenases, which participate in H2 metabolism. In the present study, H2 production by the Hox1 soluble hydrogenase and the nitrogenase were investigated. The amount of H2 evolved by the nitrogenase enzyme was much higher than the amount produced by the Hox1 hydrogenase enzyme. By comparing the H2 production by nitrogenase from five short-chain organic acids (acetate, citrate, pyruvate, succinate, formate) the highest productivity of H2 (~3 times) was observed in the presence of 4 g/l pyruvate. In this case, the pyruvate consumption was 100%, the biomass growth was equal to that of the control, therefore the produced H2 derived from pyruvate.

Hydrogenase

Hydrogenase

Since the early discovery of Prussian Blue, cyano transition metal complexes have played a fundamental role in coordination chemistry. They represent important compounds with fascinating chemical and physical properties which turn them into valuable tools for both chemists and biologists. HCN as a precursor in prebiotic chemistry has gained interest in view of its polymers being involved in the formation of amino acids, purines, and orotic acid, a biosynthetic precursor of uracil. Clearly, the rapid formation of adenine by aqueous polymerization of HCN is one of the key discoveries in these experiments. The cyanide anion is usually toxic for most aerobic organisms because of its inhibitory effects on respiratory enzymes, but as a substrate it is an important source of carbon and nitrogen for microorganisms, fungi and plants. Most interestingly, the cyanide anion is a ligand of important metal-dependent biomolecules, such as the hydrogenases and the cobalt site in vitamin B(12).

The [FeFe]-hydrogenases, although share common features when compared to other metal containing hydrogenases, clearly have independent evolutionary origins. Examples of [FeFe]-hydrogenases have been characterized in detail by biochemical and spectroscopic approaches and the high resolution structures of two examples have been determined. The active site H-cluster is a complex bridged metal assembly in which a [4Fe-4S] cubane is bridged to a 2Fe subcluster with unique non-protein ligands including carbon monoxide, cyanide, and a five carbon dithiolate. Carbon monoxide and cyanide ligands as a component of a native active metal center is a property unique to the metal containing hydrogenases and there has been considerable attention to the characterization of the H-cluster at the level of electronic structure and mechanism as well as to defining the biological means to synthesize such a unique metal cluster. The chapter describes the structural architecture of [FeFe]-hydrogenases and key spectroscopic observations that have afforded the field with a fundamental basis for understanding the relationship between structure and reactivity of the H-cluster. In addition, the results and ideas concerning the topic of H-cluster biosynthesis as an emerging and fascinating area of research, effectively reinforcing the potential linkage between iron-sulfur biochemistry to the role of iron-sulfur minerals in prebiotic chemistry and the origin of life.

[NiFe(Se)]-hydrogenases are hetero-dimeric enzymes present in many microorganisms where they catalyze the oxidation of molecular hydrogen or the reduction of protons. Like the other two types of hydrogen-metabolizing enzymes, the [FeFe]- and [Fe]-hydrogenases, [NiFe]-hydrogenases have a Fe(CO)(x) unit in their active sites that is most likely involved in hydride binding. Because of their complexity, hydrogenases require a maturation machinery that involves several gene products. They include nickel and iron transport, synthesis of CN(-) (and maybe CO), formation and insertion of a FeCO(CN(-))(2) unit in the apo form, insertion of nickel and proteolytic cleavage of a C-terminal stretch, a step that ends the maturation process. Because the active site is buried in the structure, electron and proton transfer are required between this site and the molecular surface. The former is mediated by either three or one Fe/S cluster(s) depending on the enzyme. When exposed to oxidizing conditions, such as the presence of O(2), [NiFe]-hydrogenases are inactivated. Depending on the redox state of the enzyme, exposure to oxygen results in either a partially reduced oxo species probably a (hydro)peroxo ligand between nickel and iron or a more reduced OH(-) ligand instead. Under some conditions the thiolates that coordinate the NiFe center can be modified to sulfenates. Understanding this process is of biotechnological interest for H(2) production by photosynthetic organisms.

Structural and spectroscopic studies on [Fe]-hydrogenase revealed an active site mononuclear low spin iron coordinated by the Cys176 sulfur, two CO, and the sp(2) hybridized nitrogen of a 2-pyridinol compound with back bonding properties similar to those of cyanide. Thus, [Fe]-hydrogenases are endowed with an iron-ligation pattern related to that found in the active site of [NiFe]- and [FeFe]-hydrogenases although the three hydrogenases and the enzymes involved in their posttranslational maturation have evolved independently and although CO and cyanide ligands are not found in any other metallo-enzymes. Obviously, low-spin iron complexed with thiolate(s), CO, and cyanide or a cyanide functional analogue plays an essential role in H(2) activation.