Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-ferredoxin oxidoreductase with pyruvate

Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4' amino substituent of the pyrimidine moiety of ThDP.     

Carboxy-terminal processing of the large subunit of [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757

hydA and hydB, the genes encoding the large (46-kDa) and small (13. 5-kDa) subunits of the periplasmic [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757, have been cloned and sequenced. The deduced amino acid sequence of the genes product showed complete identity to the sequence of the well-characterized [Fe] hydrogenase from the closely related species Desulfovibrio vulgaris Hildenborough (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). The data show that in addition to the well-known signal peptide preceding the NH2 terminus of the mature small subunit, the large subunit undergoes a carboxy-terminal processing involving the cleavage of a peptide of 24 residues, in agreement with the recently reported data on the three-dimensional structure of the enzyme (Y. Nicolet, C. Piras, P. Legrand, E. C. Hatchikian, and J. C. Fontecilla-Camps, Structure 7:13-23, 1999). We suggest that this C-terminal processing is involved in the export of the protein to the periplasm.     

Multiple orientations in a physiological complex: the pyruvate-ferredoxin oxidoreductase-ferredoxin system

Ferredoxin I from Desulfovibrio africanus (Da FdI) is a small acidic [4Fe-4S] cluster protein that exchanges electrons with pyruvate-ferredoxin oxidoreductase (PFOR), a key enzyme in the energy metabolism of anaerobes. The thermodynamic properties and the electron transfer between PFOR and either native or mutated FdI have been investigated by microcalorimetry and steady-state kinetics, respectively. The association constant of the PFOR-FdI complex is 3.85 x 10(5) M(-1), and the binding affinity has been found to be highly sensitive to ionic strength, suggesting the involvement of electrostatic forces in formation of the complex. Surprisingly, the punctual or combined neutralizations of carboxylate residues surrounding the [4Fe-4S] cluster slightly affect the PFOR-FdI interaction. Furthermore, hydrophobic residues around the cluster do not seem to be crucial for the PFOR-FdI system activity; however, some of them play an important role in the stability of the FeS cluster. NMR restrained docking associated with site-directed mutagenesis studies suggested the presence of various interacting sites on Da FdI. The modification of additional acidic residues at the interacting interface, generating a FdI pentamutant, evidenced at least two distinct FdI binding sites facing the distal [4Fe-4S] cluster of the PFOR. We also used a set of various small acidic partners to investigate the specificity of PFOR toward redox partners. The remarkable flexibility of the PFOR-FdI system supports the idea that the specificity of the physiological complex has probably been "sacrificed" to improve the turnover rate and thus the efficiency of bacterial electron transfer.     

Molecular dynamics study of Desulfovibrio africanus cytochrome c3 in oxidized and reduced forms

A 5-ns molecular dynamics study of a tetraheme cytochrome in fully oxidized and reduced forms was performed using the CHARMM molecular modeling program, with explicit water molecules, Langevin dynamics thermalization, Particle Mesh Ewald long-range electrostatics, and quantum mechanical determination of heme partial charges. The simulations used, as starting points, crystallographic structures of the oxidized and reduced forms of the acidic cytochrome c(3) from Desulfovibrio africanus obtained at pH 5.6. In this paper we also report structures for the two forms obtained at pH 8. In contrast to previous cytochrome c(3) dynamics simulations, our model is stable. The simulation structures agree reasonably well with the crystallographic ones, but our models show higher flexibility and the water molecules are more labile. We have compared in detail the differences between the simulated and experimental structures of the two redox states and observe that the hydration structure is highly dependent on the redox state. We have also analyzed the interaction energy terms between the hemes, the protein residues, and water. The direct electrostatic interaction between hemes is weak and nearly insensitive to the redox state, but the remaining terms are large and contribute in a complex way to the overall potential energy differences that we see between the redox states.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A278/A490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A385/A283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.     

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

Description and quantification of pteropod shell dissolution: a sensitive bioindicator of ocean acidification

Anthropogenic ocean acidification is likely to have negative effects on marine calcifying organisms, such as shelled pteropods, by promoting dissolution of aragonite shells. Study of shell dissolution requires an accurate and sensitive method for assessing shell damage. Shell dissolution was induced through incubations in CO₂‐enriched seawater for 4 and 14 days. We describe a procedure that allows the level of dissolution to be assessed and classified into three main types: Type I with partial dissolution of the prismatic layer; Type II with exposure of underlying crossed‐lamellar layer, and Type III, where crossed‐lamellar layer shows signs of dissolution. Levels of dissolution showed a good correspondence to the incubation conditions, with the most severe damage found in specimens held for 14 days in undersaturated condition (Ω ~ 0.8). This methodology enables the response of small pelagic calcifiers to acidified conditions to be detected at an early stage, thus making pteropods a valuable bioindicator of future ocean acidification.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.