Combining experiment and energy landscapes to explore anaerobic heme breakdown in multifunctional hemoproteins.

To survive, many pathogens extract heme from their host organism and break down the porphyrin scaffold to sequester the Fe2+ ion via a heme oxygenase. Recent studies have revealed that certain pathogens can anaerobically degrade heme. Our own research has shown that one such pathway proceeds via NADH-dependent heme degradation, which has been identified in a family of hemoproteins from a range of bacteria. HemS, from Yersinia enterocolitica, is the main focus of this work, along with HmuS (Yersinia pestis), ChuS (Escherichia coli) and ShuS (Shigella dysenteriae). We combine experiments, Energy Landscape Theory, and a bioinformatic investigation to place these homologues within a wider phylogenetic context. A subset of these hemoproteins are known to bind certain DNA promoter regions, suggesting not only that they can catalytically degrade heme, but that they are also involved in transcriptional modulation responding to heme flux. Many of the bacterial species responsible for these hemoproteins (including those that produce HemS, ChuS and ShuS) are known to specifically target oxygen-depleted regions of the gastrointestinal tract. A deeper understanding of anaerobic heme breakdown processes exploited by these pathogens could therefore prove useful in the development of future strategies for disease prevention.

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation.

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII (η6 -arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19 F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

Investigating the mechanisms of amylolysis of starch granules by solution-state NMR.

Starch is a prominent component of the human diet and is hydrolyzed by α-amylase post-ingestion. Probing the mechanism of this process has proven challenging, due to the intrinsic heterogeneity of individual starch granules. By means of solution-state NMR, we demonstrate that flexible polysaccharide chains protruding from the solvent-exposed surfaces of waxy rice starch granules are highly mobile and that during hydrothermal treatment, when the granules swell, the number of flexible residues on the exposed surfaces increases by a factor of 15. Moreover, we show that these flexible chains are the primary substrates for α-amylase, being cleaved in the initial stages of hydrolysis. These findings allow us to conclude that the quantity of flexible α-glucan chains protruding from the granule surface will greatly influence the rate of energy acquisition from digestion of starch.

Selective lability of ruthenium(II) arene amino acid complexes.

A series of organometallic complexes of the form [(PhH)Ru(amino acid)](+) have been synthesized using amino acids able to act as tridentate ligands. The straightforward syntheses gave enantiomerically pure complexes with two stereogenic centers due to the enantiopurity of the chelating ligands. Complexes were characterized in the solid-state and/or solution-state where the stability of the complex allowed. The propensity toward labilization of the coordinatively saturated complexes was investigated. The links between complex stability and structural features are very subtle. Nonetheless, H/D exchange rates of coordinated amino groups reveal more significant differences in reactivity linked to metallocycle ring size resulting in decreasing stability of the metallocycle as the amino acid side-chain length increases. The behavior of these systems in acid is unusual, apparently labilizing the carboxylate residue of the amino acid. This acid-catalyzed hemilability in an organometallic is relevant to the use of Ru(II) arenes in medicinal contexts due to the relatively low pH of cancerous cells.

How to make a porphyrin flip: dynamics of asymmetric porphyrin oligomers.

We present the first predictions of meso-aryl flipping pathways in porphyrin oligomers. In the context of cyclic oligoporphyrins this flipping results in a paddle rotation of each porphyrin monomer in the oligomeric ring. If the monomer porphyrin units are asymmetric, this flipping will have consequences for their supramolecular behaviour. Desymmetrisation of synthetic porphyrins leads to synthetic challenges, and hence these species are not as well studied as the more accessible, symmetric counterparts. We have both simulated and synthesized novel, desymmetrised monomeric and cyclic trimeric porphyrins and we predict that the flipping barrier for a porphyrin monomer within the trimer is 36.7 kJ mol(-1) higher than that for meso-aryl flipping in the monomer. The flipping rates estimated from Variable temperature NMR data are consistent with these results. We have also carried out a systematic investigation of how porphyrinic substituents will affect the dynamics, revealing that adding steric bulk in the right place can facilitate meso-aryl flipping. While supramolecular chemistry often focuses on highly symmetric assemblies, evolution can break molecular symmetry in subtle ways, leading to many pseudosymmetric assemblies in biology, especially protein-porphyrinic complexes that are important for energy harvesting and electron transport systems. The dynamic behaviour we have characterized can be critical for the design and function of these molecules, and hence our results will help inform future efforts in the synthesis of asymmetric porphyrinic assemblies that interact with biomolecules.

Theory and practice: bulk synthesis of C3B and its H2- and Li-storage capacity.

Previous theoretical studies of C3B have suggested that boron-doped graphite is a promising H2- and Li-storage material, with large maximum capacities. These characteristics could lead to exciting applications as a lightweight H2-storage material for automotive engines and as an anode in a new generation of batteries. However, for these applications to be realized a synthetic route to bulk C3B must be developed. Here we show the thermolysis of a single-source precursor (1,3-(BBr2)2C6H4) to produce graphitic C3B, thus allowing the characteristics of this elusive material to be tested for the first time. C3B was found to be compositionally uniform but turbostratically disordered. Contrary to theoretical expectations, the H2- and Li-storage capacities are lower than anticipated, results that can partially be explained by the disordered nature of the material. This work suggests that to model the properties of graphitic materials more realistically, the possibility of disorder must be considered.

Metastability of native proteins and the phenomenon of amyloid formation.

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.

Aberrant attachment of heme to cytochrome by the Ccm system results in a cysteine persulfide linkage.

The system I cytochrome c maturation (Ccm) apparatus has been shown to handle a wide variety of apocytochrome substrates containing the CX(n)CH heme attachment sequence, where n = 2, 3, or 4 in natural sequences. When n = 5 or 6, the apparatus also appears to handle these substrates correctly, but close inspection reveals that the resulting mature cytochromes are mixtures of species containing extra mass. We have used accurate mass spectrometry to analyze peptide digests of matured Escherichia coli cytochrome cb(562) with n = 1, 5, or 6 and shown that an extra sulfur is sometimes incorporated into the heme-protein linkage. These unprecedented, aberrant persulfide linkages may shed new light upon the mechanism of the attachment of heme to substrate apocytochrome within the Ccm complex of E. coli.

Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus.

c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b(562) to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CX(n)CH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.

Use of a fluorinated probe to quantitatively monitor amino acid binding preferences of ruthenium(ii) arene complexes.

In order to address outstanding questions about ruthenium complexes in complex biological solutions, 19F NMR spectroscopy was used to follow the binding preferences between fluorinated RuII(η6-arene)(bipyridine) complexes and protected amino acids and glutathione. Reporting what ruthenium compounds bind to in complex environments has so far been restricted to relatively qualitative methods, such as mass spectrometry and X-ray spectroscopic methods; however, quantitative information on the species present in the solution phase cannot be inferred from these techniques. Furthermore, using 1H NMR, in water, to distinguish and monitor a number of different complex RuII(η6-arene) adducts forming is challenging. Incorporating an NMR active heteroatom into ruthenium organometallic complexes provides a quantitative, diagnostic 'fingerprint' to track solution-phase behaviour and allow for unambiguous assignment of any given adduct. The resulting 19F NMR spectra show for the first time the varied, dynamic behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures. The rates of formation of the different observed species are dramatically influenced by the electronic properties at the metal, even in a closely related series of complexes in which only the electron-donating properties of the arene ligand are altered. Preference for cysteine binding is absolute: the first quantitative solution-phase evidence of such behaviour.

Designing redox metalloproteins from bottom-up and top-down perspectives.

The design of redox-active metalloproteins has been approached from two different directions. The de novo design approach has recently reached an important stage, at which structural information on several different designed metalloproteins has been obtained. This new information highlights the real challenge of this approach. The alternative approach involving re-engineering of evolved proteins has also made significant advances recently.

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562.

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.

Folates are potential ligands for ruthenium compounds in vivo.

Under physiologically relevant conditions, cis-bis(2,2'-bipyridine)dichlororuthenium(II), [cis-Ru(2,2'-bipy)2Cl2] was observed to bind to folic acid via replacement of the two chloride ligands. This binding was shown to be pH dependent and afforded diastereomers, the structures of which were determined by 1- and 2D NMR spectroscopic techniques. We propose that when studying the cytotoxicity of labile ruthenium complexes in cells, folate coordination should be considered.

Local frustration determines molecular and macroscopic helix structures.

Decorative domains force amyloid fibers to adopt spiral ribbon morphologies, as opposed to the more common twisted ribbon. We model the effect of decorating domains as a perturbation to the relative orientation of ß strands in a bilayered extended ß-sheet. The model consists of minimal energy assemblies of rigid building blocks containing two anisotropic interacting ellipsoids. The relative orientation of the ellipsoids dictates the morphology of the resulting assembly. Amyloid structures derived from experiment are consistent with our model, and we use magnets to demonstrate that the frustration principle is scale and system independent. In contrast to other models of amyloid, our model isolates the effect of frustration from the fundamental interactions between building blocks to reveal the frustration rather than dependence of morphology on the physical interactions. Consequently, amyloid is viewed as a discrete molecular version of the more general macroscopic frustrated bilayer that is exemplified by Bauhinia seedpods. The model supports the idea that the interactions arising from an arbitrary peptide sequence can support an amyloid structure if a bilayer can form first, which suggests that supplementary protein sequences, such as chaperones or decorative domains, could play a significant role in stabilizing such bilayers and therefore in selecting morphology during nucleation. Our model provides a foundation for exploring the effects of frustration on higher-order superstructural polymorphic assemblies that may exhibit complex functional behavior. Two outstanding examples are the systematic kinking of decorated fibers and the nested frustration of the Bauhinia seedpod.

Continued surprises in the cytochrome c biogenesis story.

Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein. In Gram-negative bacteria, this process is catalyzed by the Ccm (cytochrome c maturation) proteins, also called System I. The Ccm proteins are found in the bacterial inner membrane, but some (CcmE, CcmG, CcmH, and CcmI) also have soluble functional domains on the periplasmic face of the membrane. Elucidation of the mechanisms involved in the transport and relay of heme and the apocytochrome from the bacterial cytosol into the periplasm, and their subsequent reaction, has proved challenging due to the fact that most of the proteins involved are membrane-associated, but recent progress in understanding some key components has thrown up some surprises. In this Review, we discuss advances in our understanding of this process arising from a substrate's point of view and from recent structural information about individual components.

The morphology of decorated amyloid fibers is controlled by the conformation and position of the displayed protein.

Self-assembled structures capable of mediating electron transfer are an attractive scientific and technological goal. Therefore, systematic variants of SH3-Cytochrome b(562) fusion proteins were designed to make amyloid fibers displaying heme-b(562) electron transfer complexes. TEM and AFM data show that fiber morphology responds systematically to placement of b(562) within the fusion proteins. UV-vis spectroscopy shows that, for the fusion proteins under test, only half the fiber-borne b(562) binds heme with high affinity. Cofactor binding also improves the AFM imaging properties and changes the fiber morphology through changes in cytochrome conformation. Systematic observations and measurements of fiber geometry suggest that longitudinal registry of subfilaments within the fiber, mediated by the interaction and conformation of the displayed proteins and their interaction with surfaces, gives rise to the observed morphologies, including defects and kinks. Of most interest is the role of small molecule modulation of fiber structure and mechanical stability. A minimum complexity model is proposed to capture and explain the fiber morphology in the light of these results. Understanding the complex interplay between these factors will enable a fiber design that supports longitudinal electron transfer.

Metal and redox selectivity of protoporphyrin binding to the heme chaperone CcmE.

The interaction of heme with the heme chaperone CcmE is central to our understanding of cytochrome c maturation, a complex post-translational process involving at least eight proteins in many Gram-negative bacteria and plant mitochondria. We have shown previously that Escherichia coli CcmE can interact with heme non-covalently in vitro, before forming a novel covalent histidine-heme bond, in a redox-sensitive manner. The function of CcmE is to bind heme in the periplasm before transferring it to apocytochromes c. In the absence of structural information on the complex of CcmE and heme, we have further characterized it by examining the binding of the soluble domain of CcmE (CcmE') to protoporphyrins containing metals other than Fe, namely Zn-, Sn-, Co- and Mn-protoporphyrin (PPIX). CcmE' demonstrated no affinity for the Zn- or Sn-containing protoporphyrins and low affinity for Mn(ii)-PPIX. High-affinity, reversible binding was, however, observed for Co(iii)-PPIX, which was highly sensitive to oxidation state as demonstrated by release of the ligand from the chaperone on reduction; no binding to Co(ii)-PPIX was observed. The non-covalent complex of CcmE' and Co(iii)-PPIX was characterized by non-denaturing mass spectrometry. The implications of these observations for the in vivo function of CcmE are discussed.

Tuning heavy metal compounds for anti-tumor activity: is diversity the key to ruthenium's success?

This review aims to bring the reader up to date with the more recent ruthenium compounds that have been synthesized and tested for their cytotoxicity. The chemistry of these transition metal complexes will be introduced and the basic principles that govern their common behavior outlined. The recent history of established compounds within this field will be presented alongside those that now represent the cutting-edge. The inherent variety within this class of compounds will lead the reader to appreciate their diversity and pose questions as to their similarities aside from the presence of a shared metal ion. This review aims to discuss and contextualize the state-of-the-art research within the context of the speculative advancement of this developing field. There is an evident need to specify the molecular and cellular targets of these drug molecules in order to ultimately elucidate their mode or modes of action. The evidence presented herein suggests that new avenues of research require novel analytical probes and methods for tracing the fate of ruthenium complexes in cells in order to understand their very promising cytotoxic activity.