Dust is forming along the red giant branch of 47 TUC

We present additional evidence that dust is really forming along the red giant branch (RGB) of 47 Tuc at luminosities ranging from above the horizontal branch to the RGB-tip (Origlia et al. 2007). The presence of dust had been inferred from an infrared excess in the (K-8) color, with K measured from high spatial resolution ground based near-IR photometry and "8" referring to Spitzer-IRAC 8 micron photometry. We show how (K-8) is a far more sensitive diagnostic for detecting tiny circumstellar envelopes around warm giants than colors using only the Spitzer-IRAC bands, for example the (3.6-8) color used by Boyer et al. (2010). In addition, we also show high resolution HST-ACS I band images of the giant stars which have (K-8) color excess. These images clearly demonstrate that Boyer et al (2010) statement that our detections of color excess associated with stars below the RGB-tip arise from blends and artefacts is simply not valid.Comment: 12 pages, 4 figure

Semi-synthetic hydrogenases—in vitro and in vivo applications

Hydrogenases are gas processing redox enzymes central in hydrogen metabolism. The interdisciplinary nature of hydrogenase research is underscored by the development of “artificial maturation”, enabling the preparation of semi-synthetic hydrogenases through the incorporation of synthetic cofactors into a range of apo-hydrogenase hosts under in vitro and in vivo conditions. Herein, we discuss how the preparation of such semi-synthetic [FeFe]-hydrogenases has elucidated structural elements of the cofactor critical for catalysis and reactivity towards known inhibitors. It has also provided a convenient method for exploring the biodiversity of this enzyme family and thereby facilitated investigation of the role of the outer-coordination sphere in tuning the reactivity of the Hcluster. In parallel, hijacking the assembly line of the [FeFe]-hydrogenase through incorporation of synthetic precursors has provided detailed insight into the biosynthesis of the H-cluster. Moreover, it has allowed the preparation of Mn analogs of [Fe] hydrogenase

In vivo activation of an [FeFe] hydrogenase using synthetic cofactors

[FeFe] hydrogenases catalyze the reduction of protons, and oxidation of hydrogen gas, with remarkable efficiency. The reaction occurs at the H-cluster, which contains an organometallic [2Fe] subsite. The unique nature of the [2Fe] subsite makes it dependent on a specific set of maturation enzymes for its biosynthesis and incorporation into the apo-enzyme. Herein we report on how this can be circumvented, and the apo-enzyme activated in vivo by synthetic active site analogues taken up by the living cell.<p>Correction in: ENERGY &amp; ENVIRONMENTAL SCIENCE, Volume: 11, Issue: 11, Pages: 3321-3321, DOI: 10.1039/c8ee90054j</p

Discovery of novel [FeFe]-hydrogenases for biocatalytic H-2-production

A new screening method for [FeFe]-hydrogenases is described, circumventing the need for specialized expression conditions as well as protein purification for initial characterization. [FeFe]-hydrogenases catalyze the formation and oxidation of molecular hydrogen at rates exceeding 10(3) s(-1), making them highly promising for biotechnological applications. However, the discovery of novel [FeFe]-hydrogenases is slow due to their oxygen sensitivity and dependency on a structurally unique cofactor, complicating protein expression and purification. Consequently, only a very limited number have been characterized, hampering their implementation. With the purpose of increasing the throughput of [FeFe]-hydrogenase discovery, we have developed a screening method that allows for rapid identification of novel [FeFe]-hydrogenases as well as their characterization with regards to activity (activity assays and protein film electrochemistry) and spectroscopic properties (electron paramagnetic resonance and Fourier transform infrared spectroscopy). The method is based on in vivo artificial maturation of [FeFe]-hydrogenases in Escherichia coli and all procedures are performed on either whole cells or non-purified cell lysates, thereby circumventing extensive protein purification. The screening was applied on eight putative [FeFe]-hydrogenases originating from different structural sub-classes and resulted in the discovery of two new active [FeFe]-hydrogenases. The [FeFe]-hydrogenase from Solobacterium moorei shows high H-2-gas production activity, while the enzyme from Thermoanaerobacter mathranii represents a hitherto uncharacterized [FeFe]-hydrogenase sub-class. This latter enzyme is a putative sensory hydrogenase and our in vivo spectroscopy study reveals distinct differences compared to the well established H-2 producing HydA1 hydrogenase from Chlamydomonas reinhardtii

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase

Hydrogenases are among the fastest H-2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species.De två första författarna delar förstaförfattarskapet.</p

Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture

[FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely different characteristics. The M2e subclass is phylogenetically distinct from previously characterized members of this enzyme family and its biological role is unknown. It features significant differences in domain- and active site architecture, and is most closely related to the putative sensory [FeFe]-hydrogenases. Here we report the first comprehensive biochemical and spectroscopical characterization of an M2e enzyme, derived from Thermoanaerobacter mathranii. As compared to other [FeFe]-hydrogenases characterized to-date, this enzyme displays an increased H2 affinity, higher activation enthalpies for H+/H2 interconversion, and unusual reactivity towards known hydrogenase inhibitors. These properties are related to differences in active site architecture between the M2e [FeFe]-hydrogenase and â\u80\u9cprototypicalâ\u80\u9d [FeFe]-hydrogenases. Thus, this study provides new insight into the role of this subclass in hydrogen metabolism and the influence of the active site pocket on the chemistry of the H-cluster