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The deep dichotomy of archaea and bacteria is evident in many basic traits including ribosomal protein composition, membrane lipid synthesis, cell wall constituents, and flagellar composition. Here we explore that deep dichotomy further by examining the distribution of genes for the synthesis of the central carriers of one carbon units, tetrahydrofolate (H4F) and tetrahydromethanopterin (H4MPT), in bacteria and archaea. The enzymes underlying those distinct biosynthetic routes are broadly unrelated across the bacterial-archaeal divide, indicating that the corresponding pathways arose independently. That deep divergence in one carbon metabolism is mirrored in the structurally unrelated enzymes and different organic cofactors that methanogens (archaea) and acetogens (bacteria) use to perform methyl synthesis in their H4F- and H4MPT-dependent versions, respectively, of the acetyl-CoA pathway. By contrast, acetyl synthesis in the acetyl-CoA pathway - from a methyl group, CO2 and reduced ferredoxin - is simpler, uniform and conserved across acetogens and methanogens, and involves only transition metals as catalysts. The data suggest that the acetyl-CoA pathway, while being the most ancient of known CO2 assimilation pathways, reflects two phases in early evolution: an ancient phase in a geochemically confined and non-free-living universal common ancestor, in which acetyl thioester synthesis proceeded spontaneously with the help of geochemically supplied methyl groups, and a later phase that reflects the primordial divergence of the bacterial and archaeal stem groups, which independently invented genetically-encoded means to synthesize methyl groups via enzymatic reactions. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Show less

Iron-sulfur cluster proteins exhibit a range of physicochemical properties that underpin their functional diversity in biology, which includes roles in electron transfer, catalysis, and gene regulation. Transcriptional regulators that utilize iron-sulfur clusters are a growing group that exploit the redox and coordination properties of the clusters to act as sensors of environmental conditions including O2, oxidative and nitrosative stress, and metabolic nutritional status. To understand the mechanism by which a cluster detects such analytes and then generates modulation of DNA-binding affinity, we have undertaken a combined strategy of in vivo and in vitro studies of a range of regulators. In vitro studies of iron-sulfur cluster proteins are particularly challenging because of the inherent reactivity and fragility of the cluster, often necessitating strict anaerobic conditions for all manipulations. Nevertheless, and as discussed in this Account, significant progress has been made over the past decade in studies of O2-sensing by the fumarate and nitrate reduction (FNR) regulator and, more recently, nitric oxide (NO)-sensing by WhiB-like (Wbl) and FNR proteins. Escherichia coli FNR binds a [4Fe-4S] cluster under anaerobic conditions leading to a DNA-binding dimeric form. Exposure to O2 converts the cluster to a [2Fe-2S] form, leading to protein monomerization and hence loss of DNA binding ability. Spectroscopic and kinetic studies have shown that the conversion proceeds via at least two steps and involves a [3Fe-4S](1+) intermediate. The second step involves the release of two bridging sulfide ions from the cluster that, unusually, are not released into solution but rather undergo oxidation to sulfane (S(0)) subsequently forming cysteine persulfides that then coordinate the [2Fe-2S] cluster. Studies of other [4Fe-4S] cluster proteins that undergo oxidative cluster conversion indicate that persulfide formation and coordination may be more common than previously recognized. This remarkable feature suggested that the original [4Fe-4S] cluster can be restored using persulfide as the source of sulfide ion. We have demonstrated that only iron and a source of electrons are required to promote efficient conversion back from the [2Fe-2S] to the [4Fe-4S] form. We propose this as a novel in vivo repair mechanism that does not require the intervention of an iron-sulfur cluster biogenesis pathway. A number of iron-sulfur regulators have evolved to function as sensors of NO. Although it has long been known that the iron-sulfur clusters of many phylogenetically unrelated proteins are vulnerable to attack by NO, our recent studies of Wbl proteins and FNR have provided new insights into the mechanism of cluster nitrosylation, which overturn the commonly accepted view that the product is solely a mononuclear iron dinitrosyl complex (known as a DNIC). The major reaction is a rapid, multiphase process involving stepwise addition of up to eight NO molecules per [4Fe-4S] cluster. The major iron nitrosyl product is EPR silent and has optical characteristics similar to Roussin's red ester, [Fe2(NO)4(RS)2] (RRE), although a species similar to Roussin's black salt, [Fe4(NO)7(S)3](-) (RBS) cannot be ruled out. A major future challenge will be to clarify the nature of these species. Show less

Many metalloenzymes that inject and extract reducing equivalents at the beginning and the end of electron transport chains involved in chemiosmosis are suggested, through phylogenetic analysis, to have been present in the Last Universal Common Ancestor (LUCA). Their active centres are affine with the structures of minerals presumed to contribute to precipitate membranes produced on the mixing of hydrothermal solutions with the Hadean Ocean ~4 billion years ago. These mineral precipitates consist of transition element sulphides and oxides such as nickelian mackinawite ([Fe>Ni]2S2), a nickel-bearing greigite (~FeSS[Fe3NiS4]SSFe), violarite (~NiSS[Fe2Ni2S4]SSNi), a molybdenum bearing complex (~Mo(IV/VI)2Fe3S(0/2-)9) and green rust or fougerite (~[Fe(II)Fe(III)(OH)4](+)[OH](-)). They may be respectively compared with the active centres of Ni-Fe hydrogenase, carbon monoxide dehydrogenase (CODH), acetyl coenzyme-A synthase (ACS), the complex iron-sulphur molybdoenzyme (CISM) superfamily and methane monooxygenase (MMO). With the look of good catalysts - a suggestion that gathers some support from prebiotic hydrothermal experimentation - and sequestered by short peptides, they could be thought of as the original building blocks of proto-enzyme active centres. This convergence of the makeup of the LUCA-metalloenzymes with mineral structure and composition of hydrothermal precipitates adds credence to the alkaline hydrothermal (chemiosmotic) theory for the emergence of life, specifically to the possibility that the first metabolic pathway - the acetyl CoA pathway - was initially driven from either end, reductively from CO2 to CO and oxidatively and reductively from CH4 through to a methane thiol group, the two entities assembled with the help of a further thiol on a violarite cluster sequestered by peptides. By contrast, the organic coenzymes were entirely a product of the first metabolic pathways. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. Show less

Readily synthesised and functionalised di-1,2,3-triazole “click” ligands are shown to self-assemble into coordinatively saturated, quadruply stranded helical [Pd2L4](BF4)4 cages with Pd(II) ions. The cages have been fully characterised by elemental analysis, HR-ESMS, IR, 1H, 13C and DOSY NMR, DFT calculations, and in one case by X-ray crystallography. By exploiting the CuAAC “click” reaction we were able to rapidly generate a small family of di-1,2,3-triazole ligands with different core spacer units and peripheral substituents and examine how these structural modifications affected the formation of the [Pd2L4](BF4)4 cages. The use of both flexible (1,3-propyl) and rigid (1,3-phenyl) core spacer units led to the formation of discrete [Pd2L4](BF4)4 cage complexes. However, when the spacer unit of the di-1,2,3-triazole ligand was a 1,4-substituted-phenyl group steric interactions led to the formation of an oligomeric/polymeric species. By keeping the 1,3-phenyl core spacer constant the effect of altering the “click” ligands’ peripheral substituents was also examined. It was shown that ligands with alkyl, phenyl, electron-rich and electron-poor benzyl substituents all quantitatively formed [Pd2L4](BF4)4 cage complexes. The results suggest that a wide range of functionalised palladium(II) “click” cages could be rapidly generated. These novel molecules may potentially find uses in catalysis, molecular recognition and drug delivery. Show less

The quinone/semiquinone/hydroquinone triad (Q/SQ(*-)/H(2)Q) represents a class of compounds that has great importance in a wide range of biological processes. The half-cell reduction potentials of these redox couples in aqueous solutions at neutral pH, E degrees ', provide a window to understanding the thermodynamic and kinetic characteristics of this triad and their associated chemistry and biochemistry in vivo. Substituents on the quinone ring can significantly influence the electron density "on the ring" and thus modify E degrees' dramatically. E degrees' of the quinone governs the reaction of semiquinone with dioxygen to form superoxide. At near-neutral pH the pK(a)'s of the hydroquinone are outstanding indicators of the electron density in the aromatic ring of the members of these triads (electrophilicity) and thus are excellent tools to predict half-cell reduction potentials for both the one-electron and two-electron couples, which in turn allow estimates of rate constants for the reactions of these triads. For example, the higher the pK(a)'s of H(2)Q, the lower the reduction potentials and the higher the rate constants for the reaction of SQ(*-) with dioxygen to form superoxide. However, hydroquinone autoxidation is controlled by the concentration of di-ionized hydroquinone; thus, the lower the pK(a)'s the less stable H(2)Q to autoxidation. Catalysts, e.g., metals and quinone, can accelerate oxidation processes; by removing superoxide and increasing the rate of formation of quinone, superoxide dismutase can accelerate oxidation of hydroquinones and thereby increase the flux of hydrogen peroxide. The principal reactions of quinones are with nucleophiles via Michael addition, for example, with thiols and amines. The rate constants for these addition reactions are also related to E degrees'. Thus, pK(a)'s of a hydroquinone and E degrees ' are central to the chemistry of these triads. Show less

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This "local fluctuation" mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix. Show less