Alejandro Hochkoeppler

Electron Transfer Kinetics in Photosynthetic Reaction Centers Embedded in Trehalose Glasses: Trapping of Conformational Substates at Room Temperature

We report on room temperature electron transfer in the reaction center (RC) complex purified from Rhodobacter sphaeroides. The protein was embedded in trehalose-water systems of different trehalose/water ratios. This enabled us to get new insights on the relationship between RC conformational dynamics and long-range electron transfer. In particular, we measured the kinetics of electron transfer from the primary reduced quinone acceptor (Q(A)(-)) to the primary photo oxidized donor (P(+)), by time-resolved absorption spectroscopy, as a function of the matrix composition. The composition was evaluated either by weighing (liquid samples) or by near infrared spectroscopy (highly viscous or solid glasses). Deconvolution of the observed, nonexponential kinetics required a continuous spectrum of rate constants. The average rate constant ( = 8.7 s(-1) in a 28% (w/w) trehalose solution) increases smoothly by increasing the trehalose/water ratio. In solid glasses, at trehalose/water ratios > or = 97%, an abrupt increase is observed ( = 26.6 s(-1) in the driest solid sample). A dramatic broadening of the rate distribution function parallels the above sudden increase. Both effects fully revert upon rehydration of the glass. We compared the kinetics observed at room temperature in extensively dried water-trehalose matrices with the ones measured in glycerol-water mixtures at cryogenic temperatures and conclude that, in solid trehalose-water glasses, the thermal fluctuations among conformational substates are inhibited. This was inferred from the large broadening of the rate constant distribution for electron transfer obtained in solid glasses, which was due to the free energy distribution barriers having become quasi static. Accordingly, the RC relaxation from dark-adapted to light-adapted conformation, which follows primary charge separation at room temperature, is progressively hindered over the time scale of P(+)Q(A)(-) charge recombination, upon decreasing the water content. In solid trehalose-water glasses the electron transfer process resulted much more affected than in RC dried in the absence of sugar. This indicated a larger hindering of the internal dynamics in trehalose-coated RC, notwithstanding the larger amount of residual water present in comparison with samples dried in the absence of sugar.

The high potential iron-sulfur protein (HiPIP) from Rhodoferax fermentans is competent in photosynthetic electron transfer

The functional role of the High Potential Iron‐sulfur Protein (HiPIP) from the photosynthetic bacterium Rhodoferax fermentans was investigated. We demonstrated that the HiPIP increased the rate of light‐induced oxygen reduction mediated by the photosynthetic reaction center (RC); this stimulation reached half‐saturation at [HiPIP]/[RC] ca. 15. The capability of the HiPIP in delivering electrons to the reaction center of Rhodoferax fermentans was demonstrated through kinetic spectrophotometry of cytochrome c‐556 oxidation in the presence or in the absence of HiPIP. It is concluded that the HiPIP is competent in the photosynthetic electron transfer chain of Rhodoferax fermentans.

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.

Directed evolution of β‐galactosidase from Escherichia coli by mutator strains defective in the 3′→5′ exonuclease activity of DNA polymerase III

Directed evolution of Escherichia coli β‐galactosidase into variants featuring β‐glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3′→5′ exonuclease activity. β‐Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self‐transmissible episome F′128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F′128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.

HiPIP oxido-reductase activity in membranes from aerobically grown cells of the facultative phototroph Rhodoferax fermentans

The role of the periplasmically located, water‐soluble, HiPIP (high‐potential iron‐sulfur protein) in the respiratory chain of the facultative phototroph Rhodoferax fermentans has been examined. The oxidized HiPIP is reduced by succinate‐dependent respiration via the bc 1 complex, this reaction being inhibited by myxothiazol and/or stigmatellin. The reduced HiPIP can be oxidized by the membrane‐bound cytochrome oxidase, this reaction being inhibited by 0.1 mM cyanide. We conclude that aerobically grown Rf. fermentans contains a redox chain in which HiPIP mediates electron transfer between the bc 1 complex and the cb‐type cytochrome oxidase.

On the antibacterial activity of roots of Capparis spinosa L.

A decoction of Capparis spinosa L. roots, widely used in the traditional folk medicine of southern Italy, was prepared and submitted to antibacterial activity tests, which showed an interesting bacteriostatic activity on the growth of Deinococcus radiophilus. Heterocyclic compounds were also recovered from the chloroformic extract of the roots.

On the role of high-potential iron^sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs

The capability of high potential iron-sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (alpha-band at 550 nm, Em,7=+290 mV) are both involved in the electron transfer step from the bc1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c550 is more efficient than HiPIP in oxidizing the bc1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric alpha-band at 556 nm, Em,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.

On the role of cytochrome c8 in photosynthetic electron transfer of the purple non-sulfur bacterium Rhodoferax fermentans

We report on the isolation, purification and functional characterization of a soluble c-type cytochrome from light-grown cells of the purple phototroph Rhodoferax fermentans. This cytochrome is basic (pI = 8), has a molecular mass of 12 kDa, and is characterized by a midpoint reduction potential of +285 mV. Partial analysis of the N-terminus amino-acid sequence shows a high similarity with cytochromes of c8 type (formerly called Pseudomonas cytochrome c-551 type). Time-resolved spectrophotometric studies show that this cytochrome c8 reduces the tetraheme subunit of the photosynthetic reaction center, in a fast (sub-ms) and a slow (ms) phase. Competition experiments in the presence of both cytochrome c8 and high potential iron-sulfur protein (HiPIP), isolated from the same microorganism, show that cytochrome c8 oxidation is decreased upon addition of HiPIP. These observations suggest that cytochrome c8 and HiPIP might play alternative roles in the photosynthetic electron flow of Rhodoferax fermentans.

The electron transport system of the facultative phototroph Rhodoferax fermentans. II. Flash-induced oxidation of membrane-bound cytochromes c

Abstract Flash-induced oxidation of the membrane-bound c -type haems was studied in light-grown cells of Rhodoferax fermentans , a new taxon of the purple nonsulfur photosynthetic bacteria. At least three c -type cytochromes were found to rapidly ( μ s) re-reduce the photo-oxidized primary donor of the reaction centre: a first haem peaking at 556 nm with E m = 354 mV, a second haem peaking at 560 nm with E m = 294 mV, and a third haem peaking at 551 nm with E m = 79 mV. The photo-oxidized minus reduced spectrum of the primary donor was found to be very similar to those of other purple bacteria. The primary donor midpoint potential was determined (471 mV) and o -phenanthroline was found to inhibit electron transfer to the secondary acceptor, thus indicating the presence of a Q-type reaction centre. The midpoint potential of the primary quinone acceptor was also determined (13 mV). A model accounting for the post-flash redox equilibria within the RC-cytochromes c chain is presented. A tetrahaem cytochrome c is proposed to operate in Rhodoferax fermentans and its spectral and thermodynamic features are discussed in relation with other species of purple photosynthetic bacteria.