Allochromatium Vinosum Research Articles

The speciation of soluble sulphur compounds in bacterial culture fluids by X‐ray absorption near edge structure spectroscopy

Over the last decade X‐ray absorption near edge structure (XANES) spectroscopy has been used in an increasing number of microbiological studies. In addition to other applications it has served as a valuable tool for the investigation of the sulphur globules deposited intra‐ or extracellularly by certain photo‐ and chemotrophic sulphur‐oxidizing (Sox) bacteria. For XANES measurements, these deposits can easily be concentrated by filtration or sedimentation through centrifugation. However, during oxidative metabolism of reduced sulphur compounds, such as sulphide or thiosulphate, sulphur deposits are not the only intermediates formed. Soluble intermediates such as sulphite may also be produced and released into the medium. In this study, we explored the potential of XANES spectroscopy for the detection and speciation of sulphur compounds in culture supernatants of the phototrophic purple sulphur bacterium Allochromatium vinosum. More specifically, we investigated A. vinosum ΔsoxY, a strain with an in frame deletion of the soxY gene. This gene encodes an essential component of the thiosulphate‐oxidizing Sox enzyme complex. Improved sample preparation techniques developed for the ΔsoxY strain allowed for the first time not only the qualitative but also the quantitative analysis of bacterial culture supernatants by XANES spectroscopy. The results thus obtained verified and supplemented conventional HPLC analysis of soluble sulphur compounds. Sulphite and also oxidized organic sulphur compounds were shown by XANES spectroscopy to be present, some of which were not seen when standard HPLC protocols were used.

In vitro synthesis of polyhydroxyalkanoate catalyzed by class II and III PHA synthases: a useful technique for surface coatings of a hydrophobic support with PHA

AbstractIn vitro synthesis of polyhydroxyalkanoates (PHAs) on a hydrophobic support, i.e. highly oriented pyrolytic graphite (HOPG), was performed using class II PHA synthase (PhaC1Pp) from Pseudomonas putida and class III PHA synthase (PhaECAv) from Allochromatium vinosum. Using PhaC1Pp and 3‐hydroxyoctanoyl‐CoA, a poly(3‐hydroxyoctanoate) [P(3HO)] film was formed on the hydrophobic support with a thickness of a few nanometers, as revealed by atomic force microscopy (AFM). A poly(3‐hydroxybutyrate) [P(3HB)] film was also formed using PhaECAv and 3‐hydroxybutyryl‐CoA. AFM observations of the HOPG surface during P(3HB) film formation revealed that polymerized P(3HB) covered HOPG surface within the reaction time, resulting in the formation of a homogeneous ultra‐thin film. This method of ultra‐thin PHA film formation on a hydrophobic support may be applicable to the surface‐coating technique of materials with biodegradable, bioabsorbable, and biocompatible PHAs. Copyright © 2009 Society of Chemical Industry

Detection of Covalent and Noncovalent Intermediates in the Polymerization Reaction Catalyzed by a C149S Class III Polyhydroxybutyrate Synthase

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of 3-hydroxybutyryl coenzyme A (HBCoA) to PHB with a molecular mass of 1.5 MDa. The class III synthase from Allochromatium vinosum is a tetramer of PhaEPhaC (each 40 kDa). The polymerization involves covalent catalysis using C149 of PhaC with one PHB chain per PhaEC dimer. Two mechanisms for elongation have been proposed. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between C149 on each monomer. The second involves C149 and covalent and noncovalent (HB)(n)CoA intermediates. Two approaches were investigated to distinguish between these models. The first involved the wild-type (wt) PhaEC primed with sTCoA [a CoA ester of (HB)(3) in which the terminal HO group is replaced with an H] which uniformly loads the enzyme. The primed synthase was reacted with [1-(14)C]HBCoA by a rapid chemical quench method and analyzed for covalent and noncovalent intermediates. Radiolabel was found only with the protein. The second approach used C149S-PhaEC which catalyzes polymer formation at (1)/(2200) of the rate of wt-PhaEC (1.79 min(-1) vs 3900 min(-1)). C149S-PhaEC was incubated with [1-(14)C]HBCoA and chemically quenched on the minute time scale to reveal noncovalently bound [1-(14)C](HB)(2)CoA and (HB)(3)CoA as well as covalently labeled protein. Synthesized (HB)(n)CoA (n = 2 or 3) was shown to acylate PhaEC with rate constants of 1-2 min(-1), and these species were converted into polymer. Thus, the (HB)(n)CoA analogues function as kinetically and chemically competent intermediates. These results support the mechanism involving covalently and noncovalently bound intermediates.

Unexpected extracellular and intracellular sulfur species during growth of Allochromatium vinosum with reduced sulfur compounds

Before its uptake and oxidation by purple sulfur bacteria, elemental sulfur probably first has to be mobilized. To obtain more insight into this mobilization process in the phototrophic purple sulfur bacterium Allochromatium vinosum, we used HPLC analysis and X-ray absorption near-edge structure (XANES) spectroscopy for the detection and identification of sulfur compounds in culture supernatants and bacterial cells. We intended to identify soluble sulfur compounds that specifically occur during growth on elemental sulfur, and therefore compared spectra of cultures grown on sulfur with those of cultures grown on sulfide or thiosulfate. While various unexpected oxidized organic sulfur species (sulfones, C-SO(2)-C, and sulfonates, C-SO(3)(-)) were observed via XANES spectroscopy in the supernatants, we obtained evidence for the presence of monosulfane sulfonic acids inside the bacterial cells by HPLC analysis. The concentrations of the latter compounds showed a tight correlation with the content of intracellular sulfur, reaching their maximum when sulfur began to be oxidized. None of the detected sulfur compounds appeared to be a specific soluble intermediate or product of elemental sulfur mobilization. It therefore seems unlikely that mobilization of elemental sulfur by purple sulfur bacteria involves excretion of soluble sulfur-containing substances that would be able to act on substrate distant from the cells.

Increased biological hydrogen production by deletion of hydrogen-uptake system in photosynthetic bacteria

Hydrogenases are the key enzymes for the biological hydrogen production, which can be classified as H(2)-uptake hydrogenase and H(2)-production hydrogenase. The genes encoding a membrane-bound [NiFe]-hydrogenase (MBH), which is mainly responsible for hydrogen uptake, from the photosynthetic bacterium Allochromatium vinosum was cloned and sequenced. It consist of two structural genes (hydS, hydL) and two intergenic genes (isp1, isp2), which are therefore organized as hydS-isp1-isp2-hydL. This is different from the arrangement of other typical hydrogenase gene clusters. A deletion mutant-strain PhihydSL, lacking isp1, isp2, partial hydS and hydL genes, was constructed by marker-exchange mutagenesis. Under dark fermentative conditions, the hydrogen production yield by this mutant increased by 62%. The result suggests that the disruption of MBH could greatly improve the hydrogen production in the cells by decreasing the hydrogen uptake.

Altered composition of Ralstonia eutropha poly(hydroxyalkanoate) through expression of PHA synthase from Allochromatium vinosum ATCC 35206

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (<66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved alpha/beta-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB(-)4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4-5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.

Allochromatium phaeobacterium sp. nov.

A rod-shaped, phototrophic, purple sulfur bacterium was isolated in pure culture from brackish water near Bheemli, Visakhapatnam, India, in a medium that contained 2 % NaCl (w/v). Strain JA144(T) was Gram-negative and motile. It did not require salt, but tolerated up to 3 % NaCl (w/v). Intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and carotenoids that probably belonged to the rhodopinal series were present as photosynthetic pigments. Strain JA144(T) was able to grow photolithoautotrophically, photolithoheterotrophically and photoorganoheterotrophically. It did not require vitamins. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA144(T) clustered with species of the genus Allochromatium belonging to the class Gammaproteobacteria. Highest sequence similarities of strain JA144(T) were found with the type strains of Allochromatium vinosum (96.7 % similarity), Allochromatium renukae (96.3 %), Allochromatium minutissimum (95.6 %) and Allochromatium warmingii (89.2 %). Based on 16S rRNA gene sequence analysis and morphological and physiological characteristics, strain JA144(T) is sufficiently different from other Allochromatium species to represent a novel species, Allochromatium phaeobacterium sp. nov.; the type strain is JA144(T) (=JCM 14796(T)=DSM 19781(T)).

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation

Organisms using the thiosulfate-oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates ( Allochromatium vinosum), group 2 does not ( Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD) 2 which is absent in sulfur globule-forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese-like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.

Insight into the protein and solvent contributions to the reduction potentials of [4Fe–4S]2+/+ clusters: crystal structures of the Allochromatium vinosum ferredoxin variants C57A and V13G and the homologous Escherichia coli ferredoxin

The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe-4S] ferredoxin (AlvinFd) and that of the homologous ferredoxin from Escherichia coli (EcFd) have been determined at 1.05-, 1.48-, and 1.65-A resolution, respectively. The present structures combined with cyclic voltammetry studies establish clear effects of the degree of exposure of the cluster with the lowest reduction potential (cluster I) towards less negative reduction potentials (E degrees ). This is better illustrated by V13G AlvinFd (high exposure, E degrees = -594 mV) and EcFd (low exposure, E degrees = -675 mV). In C57A AlvinFd, the movement of the protein backbone, as a result of replacing the noncoordinating Cys57 by Ala, leads to a +50-mV upshift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the hydrogen-bond network involving the cluster atoms. In addition, the present structures and other previously reported accurate structures of this family of ferredoxins indicate that polar interactions of side chains and water molecules with cluster II sulfur atoms, which are absent in the environment of cluster I, are correlated to the approximately 180-250 mV difference between the reduction potentials of clusters I and II. These findings provide insight into the significant effects of subtle structural differences of the protein and solvent environment around the clusters of [4Fe-4S] ferredoxins on their electrochemical properties.

Calcium Ions Are Required for the Enhanced Thermal Stability of the Light-harvesting-Reaction Center Core Complex from Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Thermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057-1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca2+ as a cofactor. Removal of the Ca2+ from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca2+ to the Ca2+-depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Qy transition between 915 nm for the native and 880 nm for the Ca2+-depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15 degrees C higher and the enthalpy change is about 28% larger than the Ca2+-depleted LH1-RC. Substitution of the Ca2+ with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca2+ ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources.

Toward Single-Enzyme Molecule Electrochemistry: [NiFe]-Hydrogenase Protein Film Voltammetry at Nanoelectrodes

We have scaled down electrochemical assays of redox-active enzymes enabling us to study small numbers of molecules. Our approach is based on lithographically fabricated Au nanoelectrodes with dimensions down to ca. 70 x 70 nm(2). We first present a detailed characterization of the electrodes using a combination of scanning electron microscopy, cyclic voltammetry, and finite-element modeling. We then demonstrate the viability of the approach by focusing on the highly active [NiFe]-hydrogenase from Allochromatium vinosum immobilized on polymyxin-pretreated Au. Using this system, we successfully demonstrate a distinct catalytic response from less than 50 enzyme molecules. These results strongly suggest the feasibility of using bioelectrochemistry as a new tool for studying redox enzymes at the single-molecule level.

Structural and Molecular Genetic Insight into a Widespread Sulfur Oxidation Pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α2β2γ2-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.

The peripheral light-harvesting complexes from purple sulfur bacteria have different ‘ring’ sizes

The integral membrane light-harvesting (LH) proteins from purple photosynthetic bacteria form circular oligomers of an elementary unit that is composed of two very hydrophobic polypeptides, termed α and β. These apoprotein dimers are known to associate into closed circular arrays of 8, 9 and 16 α/β-mers. We report the existence of peripheral LH proteins purified from Allochromatium vinosum with two intermediate ring sizes and postulate that one is a 13 α/β-mer. This shows that LH proteins are able to form membrane rings of continuously increasing diameter from 68 to 115 Å. The presence of these new ring sizes warrants further study, as it will help to further validate the structure–function models of LH proteins currently found in the literature.

Allochromatium vinosum DsrC: Solution-State NMR Structure, Redox Properties, and Interaction with DsrEFH, a Protein Essential for Purple Sulfur Bacterial Sulfur Oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur–substrate binding/transferring protein, as well as with an electron-transfer function via thiol–disulfide interchanges.

Purification, crystallization and preliminary X-ray analysis of the membrane-bound [NiFe] hydrogenase from Allochromatium vinosum.

The membrane-bound [NiFe] hydrogenase is a unique metalloprotein that is able to catalyze the reversible oxidation of hydrogen to protons and electrons during a complex reaction cycle. The [NiFe] hydrogenase was isolated from the photosynthetic purple sulfur bacterium Allochromatium vinosum and its crystallization and preliminary X-ray analysis are reported. It was crystallized by the hanging-drop vapour-diffusion method using sodium citrate and imidazole as crystallization agents. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 205.00, b = 217.42, c = 120.44 A. X-ray diffraction data have been collected to 2.5 A resolution.

Polymyxin-Coated Au and Carbon Nanotube Electrodes for Stable [NiFe]-Hydrogenase Film Voltammetry

We report on the use of polymyxin (PM), a cyclic cationic lipodecapeptide, as an electrode modifier for studying protein film voltammetry (PFV) on Au and single-walled carbon nanotube (SWNT) electrodes. Pretreating the electrodes with PM allows for the subsequent immobilization of an active submonolayer of [NiFe]-hydrogenase from Allochromatium vinosum ( Av H2ase). Probed by cyclic voltammetry (CV), the adsorbed enzyme exhibits characteristic electrocatalytic behavior that is stable for several hours under continuous potential cycling. An unexpected feature of the immobilization procedure is that the presence of chloride ions is a prerequisite for obtaining electrocatalytic activity. Atomic force microscopy (AFM) relates the observed catalytic activity to enzymatic adsorption at the PM/Au(111) surface, and a combination of concentration-dependent CV and AFM is used to investigate the interaction between the enzyme and the PM layer.

Allochromatium renukae sp. nov.

An ovoid to rod-shaped, phototrophic, purple sulfur bacterium, designated strain JA136(T), was isolated in pure culture from brackish water near Kakinada, India, in a medium that contained 2 % NaCl (w/v). Cells were Gram-negative and motile by means of a single polar flagellum. Strain JA136(T) had no absolute salt requirement for growth but was able to tolerate up to 4 % NaCl (w/v). Intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and the carotenoid lycopene of the rhodopinal series were present as photosynthetic pigments. Strain JA136(T) was able to grow photolithoautotrophically and photolithoheterotrophically. There was no vitamin requirement for growth of strain JA136(T). Phylogenetic analysis on the basis of 16S rRNA gene sequences indicated that strain JA136(T) clustered with species of the genus Allochromatium in the class Gammaproteobacteria. Strain JA136(T) showed highest 16S rRNA gene sequence similarity with the type strains of Allochromatium vinosum (97.0 %), Allochromatium minutissimum (95.8 %) and Allochromatium warmingii (90.0 %). Based on 16S rRNA gene sequence similarity data and morphological and physiological characteristics, strain JA136(T) was sufficiently distinct from recognized Allochromatium species to be described as representing a novel species of the genus, for which the name Allochromatium renukae sp. nov. is proposed. The type strain is JA136(T) (=JCM 14262(T) =DSM 18713(T)).

Phylogeny and evolution of the family Ectothiorhodospiraceae based on comparison of 16S rRNA, cbbL and nifH gene sequences

The occurrence of genes encoding nitrogenase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was investigated in the members of the family Ectothiorhodospiraceae. This family forms a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and mostly includes photo- and chemoautotrophic halophilic and haloalkaliphilic bacteria. The cbbL gene encoding the large subunit of 'green-like' form I RubisCO was found in all strains, except the type strains of Alkalispirillum mobile and Arhodomonas aquaeolei. The nifH gene encoding nitrogenase reductase was present in all investigated species of the phototrophic genera Ectothiorhodospira, Halorhodospira and Thiorhodospira, but not of the genus Ectothiorhodosinus. Unexpectedly, nifH fragments were also obtained for the chemotrophic species Thioalkalispira microaerophila and Alkalilimnicola halodurans, for which diazotrophic potential has not previously been assumed. The cbbL-, nifH- and 16S rRNA gene-based trees were not highly congruent in their branching patterns since, in the 'RubisCO' and 'nitrogenase' trees, representatives of the Ectothiorhodospiraceae are divided in a number of broadly distributed clusters and branches. However, the data obtained may be regarded as evidence of the monophyletic origin of the cbbL and nifH genes in most species within the family Ectothiorhodospiraceae and mainly corresponded to the current taxonomic structure of this family. The cbbL phylogeny of the chemolithoautotrophic sulfur-oxidizers Thioalkalivibrio nitratireducens and Thioalkalivibrio paradoxus and the nitrifier Nitrococcus mobilis deviated significantly from the 16S-rRNA gene-based phylogeny. These species clustered with one of the duplicated cbbL genes of the purple sulfur bacterium Allochromatium vinosum, a member of the family Chromatiaceae.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DsrEFH fromAllochromatium vinosum

In purple sulfur bacteria, the proteins encoded by dsr genes play an essential role in the oxidation of intracellular sulfur, which is an obligate intermediate during the oxidation of sulfide and thiosulfate. One such gene product, DsrEFH from Allochromatium vinosum, has been cloned, expressed, purified and crystallized. Synchrotron data were collected to 2.5 A from a crystal of selenomethionine-substituted DsrEFH. The crystal belongs to the primitive monoclinic space group P2(1), with unit-cell parameters a = 56.6, b = 183.1, c = 107.8 A, beta = 99.6 degrees. A full structure determination is under way in order to provide insight into the structure-function relationships of this protein.

Ligand-induced monomerization of Allochromatium vinosum cytochrome c′ studied using native mass spectrometry and fluorescence resonance energy transfer

Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.