Herrick Baltscheffsky

Inorganic Pyrophosphate: Formation in Bacterial Photophosphorylation

Inorganic pyrophosphate is identified as the major product of photophosphorylation by isolated chromatophores from Rhodospirillum rubrum in the absence of added nucleotides.

H+ -PPases: a tightly membrane-bound family.

The earliest known H+‐PPase (proton‐pumping inorganic pyrophosphatase), the integrally membrane‐bound H+‐PPi synthase (proton‐pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H+‐ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+‐PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton‐pumping enzymes. The bacterial H+‐PPi synthase and two algal vacuolar H+‐PPases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+‐PPases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+‐PPases are reviewed and compared with H+‐ATPases and soluble PPases.

H+-PPases: yesterday, today and tomorrow.

Suggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy‐rich phosphate donor in biological energy conversion, were followed in the mid‐1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membrane‐bound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy‐requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H+‐PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H+‐PPases differing in K+ sensitivity were established. Over 200 H+‐PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H+‐PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high‐molecular‐weight polyphosphates (HMW‐polyP). Here we will focus attention on the H+‐PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures. IUBMB Life, 59: 76‐83, 2007

The light-induced, reversible pH change in chromatophores from Rhodospirillum rubrum☆

Abstract The light-induced pH change which has been demonstrated in chromatophores from Rhodospirillum rubrum is sensitive to low concentrations of valinomycin, gramicidin, and oligomycin. As is the case in suspensions of animal mitochondria, valinomycin in contrast to gramicidin requires addition of K + for its action. In contrast to the situation with mitochondria, valinomycin differs drastically from gramidicin in its gross action on energy-requiring ion movement in chromatophores. Thus valinomycin strongly stimulates, whereas gramicidin inhibits, the light-induced pH change in chromatophores. Both agents stimulate the “off” reaction. Oligomycin slightly stimulates the rate and increases the extent of the pH change, but the “off” reaction is less affected. The chromatophores have been found to constitute (at the present time) a unique experimental system for investigating the energy-requiring ion movement by using light as an energy source, which can be rapidly varied between the extreme levels of excess and zero and being sensitive to low concentrations of agents such as valinomycin and gramicidin, which specifically interfere with energy-requiring ion movement processes.

Flavin nucleotides and light-induced phosphorylation in cell-free extracts of Rhodospirillum rubrum.

Abstract Light-induced phosphorylation (LIP) in extracts of the photosynthetic bacterium Rhodospirillum rubrum can be stimulated by addition of FAD, but not by addition of FMN. Atebrin, a flavin antagonist, strongly inhibits LIP. This inhibition can be completely overcome with FAD but only partially with FMN. The inhibition of LIP by atebrin is equally strong in the presence of phenazine methosulfate, which stimulates LIP, as in its absence. Thus, in contrast to the case when antimycin A or 2- n -heptyl-4-hydroxyquinoline-N-oxide (HOQNO) are used as inhibitors, phenazine methosulfate does not provide a “by-pass” around the point of action of atebrin. Antimycin A and HOQNO inhibit LIP almost completely both in the presence and in the absence of added FAD. This indicates that any electrons passing through FAD also pass through the site which is inhibited by antimycin A and HOQNO. It is suggested that FAD is an electron transport carrier in LIP in cell-free extracts of Rhodospirillum rubrum .

Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum

Acetylene reduction catalyzed by nitrogenase from Rhodospirillum rubrum has low activity and exhibits a lag phase. The activity can be increased by the addition of a chromatophore membrane component and the lag eliminated by preincubation with this component, which can be solubilized from chromatophores by treatment with NaCl. It is both trypsin- and oxygen-sensitive. Titration of the membrane component with nitrogenase and vice versa shows a saturation point. The membrane component interacts specifically with the Fe protein of nitrogenase, the interaction being ATP- and Mg2+-dependent.

H+-proton-pumping inorganic pyrophosphatase: a tightly membrane-bound family

The earliest known H+‐proton‐pumping inorganic pyrophosphatase, the integrally membrane‐bound H+‐proton‐pumping inorganic pyrophosphate synthase from Rhodospirillum rubrum, is still the only alternative to H+‐ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+‐proton‐pumping inorganic pyrophosphatase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton‐pumping enzymes. The bacterial H+‐proton‐pumping inorganic pyrophosphate synthase and two algal vacuolar H+‐proton‐pumping inorganic pyrophosphatases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+‐proton‐pumping inorganic pyrophosphatases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+‐proton‐pumping inorganic pyrophosphatases are reviewed and compared with H+‐ATPases and soluble proton‐pumping inorganic pyrophosphatases.

Isolation and properties of succinate dehydrogenase from Rhodospirillum rubrum

Abstract Succinate dehydrogenase has been solubilized from R. rubrum chromatophores with the use of chaotropic agents, and purified approximately 80-fold. The preparation (SDr) contains 8 g-atoms of iron per mole of flavin, and has a turnover number of approximately 4000 (moles succinate oxidized by ferricyanide or phenazine methosulfate/mole of flavin/min at 38 °C). Its absorption and EPR spectra are similar to those of bovine heart succinate dehydrogenase. SDr can cross-interact with the bovine heart electron-transport system (alkali-inactivated ETP) and reconstitute succinoxidase activity with an efficiency comparable to the reconstitution activity of purified bovine heart succinate dehydrogenase. Preliminary results suggest that SDr has a molecular weight of approximately 85,000, and that it is composed of a flavoprotein subunit with a molecular weight of approximately 60,000, plus a second subunit (possibly an iron-sulfur protein) with a molecular weight of approximately 25,000.

Evidence for two phosphorylation sites in bacterial cyclic photophosphorylation

The results from studies of cyclic photophosphorylation in isolated chromatophores from Rhodospirillum rubrum with the uncoupling agent valinomycin provide evidence for the existence of two phosphorylation sites in the operating cyclic electron transport chain. Low concentrations of valinomycin uncouple the phosphorylation which is linked to one of these sites, but does not uncouple the phosphorylation which is linked to the other site. When the electron transport is inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide at a specific site and the electron carrier phenazine methosulfate is employed to “by-pass” the inhibition, only the phosphorylation reactions which are insensitive to valinomycin are active. Thus, when the electrons are transported along the “phenazine methosulfate-pathway”, the “by-passed” part of the electron transport chain contains the phosphorylation site to which the valinomycin-sensitive phosphorylation is coupled.

Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae

We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.