Gas-phase and heat-exchange effects on the ignition of high- and low-exothermicity porous solids subject to constant heating

This article investigates the ignition of low-exothermicity reactive porous solids exposed to a maintained source of heat (hotspot), without oxygen limitation. The gas flow within the solid, particularly in response to pressure gradients (Darcy’s law), is accounted for. Numerical experiments related to the ignition of low-exothermicity porous materials are presented. Gas and solid products of reaction are included. The first stage of the paper examines the (pseudo-homogeneous) assumption of a single temperature for both phases, amounting to an infinite rate of heat exchange between the two. Isolating the effect of gas production and flow in this manner, the effect of each on the ignition time is studied. In such cases, ignition is conveniently defined by the birth of a self-sustained combustion wave. It is found that gas production decreases the ignition time, compared to equivalent systems in which the gas-dynamic problem is effectively neglected. The reason for this is quite simple; the smaller heat capacity of the gas allows the overall temperature to attain a higher value in a similar time, and so speeds up the ignition process. Next, numerical results using a two-temperature (heterogeneous) model, allowing for local heat exchange between the phases, are presented. The pseudo-homogeneous results are recovered in the limit of infinite heat exchange. For a finite value of heat exchange, the ignition time is lower when compared to the single-temperature limit, decreasing as the rate of heat exchange decreases. However, the decrease is only mild, of the order of a few percent, indicating that the pseudo-homogeneous model is in fact a rather good approximation, at least for a constant heat-exchange rate. The relationships between the ignition time and a number of physico-chemical parameters of the system are also investigated

The effect of oxygen starvation on ignition phenomena in a reactive solid containing a hot-spot

In this paper, we explore the effect of oxygen supply on the conditions necessary to sustain a self-propagating front from a spherical source of heat embedded in a much larger volume of solid. The ignition characteristics for a spherical hot-spot are investigated, where the reaction is limited by oxygen, that is, reactant + oxygen ? product. It is found that over a wide range of realistic oxygen supply levels, constant heating of the solid by the hot-spot results in a self-propagating combustion front above a certain critical hot-spot power; this is clearly an important issue for industries in which hazard prevention is important. The ignition event leading to the formation of this combustion wave involves an extremely sensitive balance between the heat generated by the chemical reaction and the depletion of the reactant. As a result, for small hot-spot radii and infinite oxygen supply, not only is there a critical power above which a self-sustained combustion front is initiated there also exists a power beyond which no front is formed, before a second higher critical power is found. The plot of critical power against hot-spot radius thus takes on a Z-shape appearance. The corresponding shape for the oxygen-limited reaction is qualitatively the same when the ratio of solid thermal diffusion to oxygen mass diffusion (N) is small and we establish critical conditions for the initiation of a self-sustained combustion front in that case. As N gets larger, while still below unity, we show that the Z-shape flattens out. At still larger values of N, the supercritical behaviour becomes increasingly difficult to define and is supplanted by burning that depends more uniformly on power. In other words, the transition from slow burning to complete combustion seen at small values of N for some critical power disappears. Even higher values of N lead to less solid burning at fixed values of power

Five Glutamic Acid Residues in the C-Terminal Domain of the ChlD Subunit Play a Major Role in Conferring Mg2+ Cooperativity upon Magnesium Chelatase

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg2+ ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway

Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

Quantifying cellular components is a basic and important step for understanding how a cell works, how it responds to environmental changes, and for re-engineering cells to produce valuable metabolites and increased biomass. We quantified proteins in the model cyanobacterium Synechocystis sp. PCC 6803 given the general importance of cyanobacteria for global photosynthesis, for synthetic biology and biotechnology research, and their ancestral relationship to the chloroplasts of plants. Four mass spectrometry methods were used to quantify cellular components involved in the biosynthesis of chlorophyll, carotenoid and bilin pigments, membrane assembly, the light reactions of photosynthesis, fixation of carbon dioxide and nitrogen, and hydrogen and sulfur metabolism. Components of biosynthetic pathways, such as those for chlorophyll or for photosystem II assembly, range between 1000 and 10,000 copies per cell, but can be tenfold higher for CO2 fixation enzymes. The most abundant subunits are those for photosystem I, with around 100,000 copies per cell, approximately 2 to fivefold higher than for photosystem II and ATP synthase, and 5–20 fold more than for the cytochrome b6f complex. Disparities between numbers of pathway enzymes, between components of electron transfer chains, and between subunits within complexes indicate possible control points for biosynthetic processes, bioenergetic reactions and for the assembly of multisubunit complexes

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2- dependent insertion of a Mg2+ ion into protoporphyin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH. Quantitative analysis using microscale thermophoresis (MST) showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical crosslinking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. Our work, showing the interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase, will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH

X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond

The active site of magnesium chelatase

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons