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Research conducted on the ability of Rhodospirillum rubrum to reduce selenite and to detoxificate elemental selenium is presented. Results include a demonstration of the detoxification process.

An investigation of solubilization organelle and plasma membranes by diheptanoylphosphatidylcholine (DHPC) indicates that the DHPC functions as a mild detergent and is capable of complete solubilization. The DHPC exhibits a high level of dispersing power, which does not affect the structure of the native protein. The enzymatic activity remains the same even after the process of solubilization by interacting with the lipid membranes and not by reacting with the proteins found in the membranes.

This opinion review explores the microbiology of tellurite, TeO 3 2- and selenite, SeO 3 2- oxyanions, two similar Group 16 chalcogen elements, but with slightly different physicochemical properties that lead to intriguing biological differences. Selenium, Se, is a required trace element compared to tellurium, Te, which is not. Here, the challenges around understanding the uptake transport mechanisms of these anions, as reflected in the model organisms used by different groups, are described. This leads to a discussion around how these oxyanions are subsequently reduced to nanomaterials, which mechanistically, has controversies between ideas around the molecule chemistry, chemical reactions involving reduced glutathione and reactive oxygen species (ROS) production along with the bioenergetics at the membrane versus the cytoplasm. Of particular interest is the linkage of glutathione and thioredoxin chemistry from the cytoplasm through the membrane electron transport chain (ETC) system/quinones to the periplasm. Throughout the opinion review we identify open and unanswered questions about the microbial physiology under selenite and tellurite exposure. Thus, demonstrating how far we have come, yet the exciting research directions that are still possible. The review is written in a conversational manner from three long-term researchers in the field, through which to play homage to the late Professor Claudio Vásquez., (© 2022. The Author(s).)

Background In many works, the chemical composition of bacterially-produced elemental selenium nanoparticles (Se 0 -nanoparticles) was investigated using electron dispersive X-ray analysis. The results suggest that these particles should be associated with organic compounds. However, a complete analysis of their chemical composition is still missing. Aiming at identifying organic compounds associated with the Se 0 -nanoparticles produced by the purple phototrophic bacteria Rhodospirillum rubrum and Rhodobacter capsulatus (α group of the proteobacteria), we used MALDI-TOF spectrometry.Results This technic revealed that numerous signals obtained from particles produced by both species of bacteria were from metabolites of the photosynthetic system. Furthermore, not only bacteriochlorophyll a, bacteriopheophytin a, and bacteriopheophorbide a, which are known to accumulate in stationary phase cultures of these bacteria grown phototrophically in the absence of selenite, were identified. The particles were also associated with intermediary metabolites of the bacteriochlorophyll a biosynthesis pathway such as protoporphyrin IX, protoporphyrin IX monomethyl ester, bacteriochlorophyllide a and, most likely, Mg-protoporphyrin IX-monomethyl ester, as well as with oxidation products of the substrates of protochlorophyllide reductase and chlorin reductase.Conclusion Accumulation of intermediary metabolites of the bacteriochlorophyll biosynthesis pathway in these purple phototrophic bacteria was attributed to inhibition of oxygen-sensitive enzymes involved in this pathway. Consistent with this interpretation it has been reported that these bacteria reduce selenite intracellularly, that they contain high levels of glutathione and that the reduction of selenite with glutathione is a very fast reaction accompanied by the production of reactive oxygen species. As many enzymes involved in the biosynthesis of bacteriochlorophyll contain [Fe-S] clusters in their active site, which are known to be degraded in the presence of reactive oxygen species as well as in the presence of molecular oxygen, we concluded that the substrates of these enzymes accumulate in cells during selenite reduction.Association of metabolites of bacteriochlorophyll biosynthesis and degradation with the Se 0 -nanoparticles produced by Rhodospirillum rubrum and Rhodobacter capsulatus is proposed to result from coating of the nanoparticles with the intracytoplasmic membrane of these bacteria, where the photochemical apparatus is concentrated.

Various enzymic systems, such as nitrite reductase, sulfite reductase and glutathione reductase, have been proposed for, or suspected to be involved in, the reduction of selenite in bacteria. As alphaproteobacteria have been shown to be highly tolerant to transition metal oxyanions, it seemed interesting to investigate the hypothetical involvement of these different enzymes in the reduction of selenite in the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus. The hypothetical involvement of nitrite reductase and sulfite reductase in the reduction of selenite in these bacteria was investigated by analysing the effects of nitrite and sulfite amendments on the growth and kinetics of selenite reduction. The reduction of selenite was not concomitant with that of either sulfite or nitrite in Rs. rubrum, suggesting that the reduction pathways operate independently. In Rb. capsulatus, strong interactions were observed between the nitrite reduction and selenite reduction pathways. However, in both organisms, selenite reduction took place during both the growth phase and the stationary phase, indicating that selenite metabolism is constitutively expressed. In contrast, neither nitrite nor sulfite was transformed during stationary phase, suggesting that the metabolism of both ions is induced, which implies that identical reduction pathways for selenite and nitrite or selenite and sulfite are excluded. Buthionine sulfoximine (BSO, S-n-butyl homocysteine sulfoximine), a specific inhibitor of glutathione synthesis, was used to depress the intracellular glutathione level. In stationary-phase cultures of both Rs. rubrum and Rb. capsulatus amended with BSO, the rate of reduction of selenite was slowed, indicating that glutathione may be involved in the dissimilatory reduction of selenite in these organisms. The analysis of the headspace gases of the cultures indicated that the synthesis of methylated selenium compounds was prevented in the presence of 3.0 mM BSO in both organisms, implying that glutathione is also involved in the transformation of selenite to volatile selenium compounds.

Various mechanisms have been proposed to explain the biological dissimilatory reduction of selenite (SeO3(2-)) to elemental selenium (Se(o)), although none is without controversy. Glutathione, the most abundant thiol in the eukaryotic cells, the cyanobacteria, and the alpha, beta, and gamma groups of the proteobacteria, has long been suspected to be involved in selenium metabolism. Experiments with the phototrophic alpha proteobacterium Rhodospirillum rubrum showed that the rate of selenite reduction was decreased when bacteria synthesized lower than normal levels of glutathione, and in Rhodobacter sphaeroides and Escherichia coli the reaction was reported to induce glutathione reductase. In the latter organism superoxide dismutase was also induced in cells grown in the presence of selenite, indicating that superoxide anions (O2-) were produced. These observations led us to investigate the abiotic (chemical) reduction of selenite by glutathione and to compare the features of this reaction with those of the reaction mediated by R. rubrum and E. coli. Our findings imply that selenite was first reduced to selenodiglutathione, which reached its maximum concentration within the 1st min of the reaction. Formation of selenodiglutathione was paralleled by a rapid reduction of cytochrome c, a known oxidant for superoxide anions. Cytochrome c reduction was inhibited by superoxide dismutase, indicating that O2- was the source of electrons for the reduction. These results demonstrated that superoxide was produced in the abiotic reduction of selenite with glutathione, thus lending support to the hypothesis that glutathione may be involved in the reaction mediated by R. rubrum and E. coli. The second phase of the reaction, which led to the formation of elemental selenium (Se(o)), developed more slowly. Se(o) precipitation reached a maximum within 2 h after the beginning of the reaction. Secondary reactions leading to the degradation of the superoxide significantly decreased the yield of Se(o) in the abiotic reaction compared with that of the bacterially mediated selenite reduction. Abiotically formed selenium particles showed the same characteristic orange-red color, spherical structure, and size as particles produced by R. rubrum, again providing support for the hypothesis that glutathione is involved in the reduction of selenite to elemental selenium in this organism.

To probe the catalytic mechanism of human purine nucleoside phosphorylase (PNP), 13 active-site mutants were constructed and characterized by steady-state kinetics. In addition, microtiter plate assays were developed for both the phosphorolytic and synthetic reactions and used to determine the kinetic parameters of each mutant. Mutations in the purine binding site exhibited the largest effects on enzymatic activity with the Asn243Ala mutant resulting in a 1000-fold decrease in the kcat for inosine phosphorolysis. This result in combination with the crystallographic location of the Asn243 side chain suggested a potential transition state (TS) structure involving hydrogen bond donation by the carboxamido group of Asn243 to N7 of the purine base. Analogous to the oxyanion hole of serine proteases, this hydrogen bond was predicted to aid catalysis by preferentially stabilizing the TS as a consequence of the increase in negative charge on N7 that occurs during glycosidic bond cleavage and the associated increase in the N7-Asn243 hydrogen bond strength. Two residues in the phosphate binding site, namely His86 and Glu89, were also predicted to be catalytically important based on their alignment with phosphate in the X-ray structure and the 10-25-fold reduction in catalytic activity for the His86Ala and Glu89Ala mutants. In contrast, catalytic efficiencies for the Tyr88Phe and Lys244Ala mutants were comparable with wild-type, indicating that the hydrogen bonds predicted in the initial X-ray structure of PNP [Ealick, S. E., et al. (1990) J. Biol. Chem. 265, 1812-1820] were not essential for catalysis. These results provided the foundation for studies reported in the ensuing two manuscripts focused on the PNP catalytic mechanism [Erion, M. D., et al. (1997) Biochemistry 36, 11735-11748] and the use of mutagenesis to reverse the PNP substrate specificity from 6-oxopurines to 6-aminopurines [Stoeckler, J. D., et al. (1997) Biochemistry 36, 11749-11756].

Chromatophores from Rhodospirillum rubrum were solubilized using the detergent 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC). The solubilization curves are sigmoidal reaching a plateau at a detergent/protein ratio of 2-3 μmol/mg corresponding to 75-90% solubilized protein. The BChl-binding proteins are stable over a large range of DHPC/protein ratios. A complex of BChl-binding proteins containing both LHI- and RC-polypeptides (LHI-RC-complex) was purified using a two step procedure. RC photochemical activity as well as absorption and near-IR CD spectra showed the complex to be active and stable after purification in presence of DHPC.