A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster

Exploring the integration of social media within integrated marketing communication frameworks: perspectives of services marketers

Purpose – At present no frameworks exist for services marketers to incorporate social media (SM) within marketing communications planning. The majority of integrated marketing communications (IMC) frameworks were developed prior to the development of the widespread use of digital and SM for information seeking, sales and service. The purpose of this paper is to investigate this issue for services marketers specifically as they differ from FMCG, industrial and durable marketers in terms of marketing messages, branding, media and channels. Furthermore, as they are less reliant on outsourced sale channels they have more potential than other industries to integrate social and digital media to build awareness, brands and sales. Design/methodology/approach – Depth interviews were conducted with eight senior services marketing executives to identify the impact of SM on marketing communications planning, implementation and measurement. Findings – The findings revealed that the unique characteristics of SM (such as interactivity and individualisation, integration of communication and distribution channels, immediacy and information collection) impact traditional marketing communications frameworks. These impacts manifested in 12 modifications specific to services and SM to traditional generic IMC frameworks encompassed by the themes of reach, service channel, word-of-mouth advocacy, consumer generated messages, listening and behavioural measurement. Practical implications – The rapidly evolving nature of SM means senior services marketers need to educate organisational stakeholders regarding implementation issues, which may be a barrier to effective integration of SM within marketing communications. Originality/value – With digital marketing communications budgets reaching 30 per cent within some organisations, it is timely to put forward a marketing communication decision-making framework that first incorporates SM and second is suitable for services marketers

Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN− Ligands Derive from Tyrosine

[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN−) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN− ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

BACKGROUND: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. PRINCIPAL FINDINGS: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms. SIGNIFICANCE: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments

Mapping hotel brand positioning and competitive landscapes by text-mining user-generated content

YesThis study uncovers hotel brand positioning and competitive landscape mapping by text-mining user-generated content (UGC). Rather than relying on a single dimension of consumer evaluation, the current study detects brand attributes by using both customer preferences as well as perceptual performance to develop meaningful insights. For this, the study combines content analysis and repertory grid analysis (RGA) to answer three key research issues. 111,986 hotel reviews from two biggest Chinese cities are used to explore and visualize the competitive landscape of six selected hotel brands across three hotel categories. Findings from the study will not only advance the existing literature on brand positioning and competitive landscape mapping but also help practitioners in developing brand positioning strategies to fight competitors within and across hotel categories

Mechanistic and structural characterisation of HydG catalysed L-tyrosine cleavage

The S-adenosyl-L-methionine (AdoMet) dependent enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase cofactor assembly. It catalyses L-tyrosine cleavage to yield the cofactor cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve the understanding of the roles of each of these iron?sulfur clusters in catalysis, HydG variants lacking either the N- or C-terminal cluster have been generated and examined using spectroscopic and kinetic methods. Quantification of coordinated iron, UV?Vis spectroscopy and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant which cannot coordinate the radical AdoMet cluster was used to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (?CTD) harbouring only the N-terminal cluster demonstrates that both clusters have similar UV?Vis and EPR spectral properties, but that AdoMet binding and cleavage can only occur at the N-terminal radical AdoMet cluster. The latter observation was also confirmed by initial structural characterisation of a thermostable Thermoanaerobacter italicus HydG. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, Michaelis?Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ?CTD Clostridium acetobutylicum HydG were determined and demonstrate that these C-terminal modifications do not strongly affect the affinity for AdoMet, but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. A definite tyrosine binding site could unfortunately not be established in the Thermoanaerobacter italicus HydG crystal structure. Further kinetic characterisation of the above Clostridium acetobutylicum HydG mutants suggests that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine derived intermediate to cyanide and CO