The golden age of biochemical research in photosynthesis

The perspectives and enthusiasms recorded in this review describe the events I witnessed and, in small ways, contributed to. Two great rewards emerged from my experiences – the pleasure of doing experiments and the great wealth of friendships with students and colleagues. As a graduate student, phenomena appeared at the bench before me which clarified the coupling of electron transport to ATP synthesis. My first PhD graduate student measured concentrations of pyridine nucleotides in chloroplasts and his results have been often confirmed and well used. All of the many graduate students who followed contributed to our understanding of photosynthesis. I have taken much pleasure from documenting the details of photosynthetic phosphorylation and electron transport in cyanobacteria. Studies of the `c' type cytochromes in these organisms continue to fascinate me. My experiences in government in its efforts to promote research are unusual, perhaps unique. A rare event outside the laboratory – a natural bloom of cyanobacteria – stimulated new thoughts and special opportunities for laboratory science. Photosynthesis seems magisterial in its shaping of our planet and its biology and in the details of its cleverness that were revealed in the time of my witness. This revised version was published online in June 2006 with corrections to the Cover Date.     

Contributions of Henrik Lundegårdh

Henrik Lundegårdh made major contributions in the field of ecology and plant physiology from 1912 to 1969. His early work at Hallands Väderö in the Kattegat pioneered quantitative approaches to plant ecology and laid the understanding of carbon dioxide exchange in natural communities which is still useful today in global carbon accounting. Very early on in this work he invented the flame photometer. In trying to understand salt respiration of plants, he started to formulate hypotheses for the relationship between respiration and ion movement, including protons, hypotheses that were forerunners to the Chemiosmotic Hypothesis of Peter Mitchell. Necessarily, this involved work on plant cytochromes. He invented several early recording spectrophotometers and made many early discoveries in the field of plant cytochromes, including the photo-oxidation of cytochrome f in photosynthesis.     

Feeding fried oil changes antioxidant and fatty acid pattern of rat and affects rat liver mitochondrial respiratory chain components

Fat frying is a popular food preparation method but several components like antioxidant vitamins could be lost due to oxidation and some others with toxic effects could appear. Because of such large consumption of frying oils, the effect of high temperatures on the oils is of major concern both for product quality and nutrition, taking into account that dietary fat source deeply influences several biochemical parameters, especially of mitochondrial membranes. Virgin olive oil possesses specific features for modulating the damages occurred by endogenous and exogenous oxidative stress being particularly rich in antioxidant molecules. We evaluated the extent of modifications suffered by virgin olive oil following a short-time deep fat frying procedure: vitamin E and phenolic compound as well as total antioxidant capacity (measured by ESR) decreased, while polar compounds increased. The intake of such an altered oil mainly affected the hydroperoxide and TBARS contents of mitochondrial membranes which were enhanced after the dietary treatments. Also, several mitochondrial respiratory chain components (Coenzyme Q, cytochrome b, c + c ₁, and a + a ₃) were affected.     

Ageing-related tissue-specific alterations in mitochondrial composition and function are modulated by dietary fat type in the rat

This study investigated the way in which feeding rats with two fat sources (olive or sunflower oils) affected electron-transport components and function of mitotic (liver) and postmitotic (heart and skeletal muscle) tissues during ageing. Rats adapted the mitochondrial-membrane-lipid profile to dietary fat throughout the study, suggesting that the benefits to eat either of the two fats might be maintained lifelong. Liver was more resistant to dietary changes and ageing than heart and skeletal muscle, which showed higher levels of coenzyme Q, cytochrome b, and cytochrome a + a ₃ with ageing and lower cytochrome c oxidase and complex IV turnover. Dietary fat differentially modulated the response of tissues during ageing, with sunflower oil leading to the highest levels of coenzyme Q and cytochromes b and a + a ₃. Since high levels of cytochrome b have been related to increased age, it could be hypothesized that olive oil could lead to less aged mitochondria.     

Studies of the Electron Transport Chain of the Euryarcheon Halobacterium salinarum: Indications for a Type II NADH Dehydrogenase and a Complex III Analog

The components involved in the respiratory system of the euryarcheon Halobacterium salinarum were investigated by spectroscopic and polarographic techniques. Previous results about the cytochrome composition could be verified. However, under low oxygen tension, the expression of a d-type cytochrome was detected. Membranes exerted an NADH– and succinate–cytochrome-c oxidoreductase as well as an NADH and succinate oxidase activity. These activities could be blocked by the following inhibitors: 7-jodocarboxylic acid, giving evidence for the presence of a type II NADH dehydrogenase, antimycin A, and myxothiazol, indicating the presence of a complex III analog, and the typical succinate dehydrogenase (SDH) and terminal oxidase inhibitors. Complex I inhibitors like rotenone and annonine were inactive, clearly excluding the presence of a coupled NADH dehydrogenase. In addition, no [Fe-S] resonances in the region of the NADH dehydrogenase (NDH) clusters could be observed after NADH addition. One of the terminal oxidases could be shown to act as a cytochrome-c oxidase with a K _m value of 37 M and an activation energy of 23.7 kJ/mol. The relative molecular mass of the endogenous c-type cytochrome could be determined as 14.1 kD. The complex III analog could be enriched after detergent extraction with Triton X-100 and hydroxylapatite (HTP) chromatography. The partially purified complex contained a Rieske iron–sulfur cluster, b- and c-type cytochromes, and was catalytically active in the decylubiquinone–cytochrome-c oxidoreductase assay.     

The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum

We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3 end of cycH were termed cycJ, cycK and cycL. A deletion mutant (cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an M_r of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.     

Actinomycete cytochromes P‐450 involved in oxidative metabolism: Biochemistry and molecular biology∗

The diverse metabolic capability of actinomycetes, together with their widespread presence in terrestrial and aquatic environments, has rendered them instrumental in the breakdown of both man‐made and natural chemicals and in the recycling of carbon in nature. Actinomycetes are also well known for their ability to synthesize a wide variety of novel chemicals of pharmaceutical and industrial value. Cytochromes P‐450, a class of oxidative enzymes found in all organisms, play a central role in both biosynthetic and biodegradative reactions performed by actinomycetes. Herein, we describe recent studies of actinomycetes cytochromes P‐450 made possible by advances realized in biochemistry and molecular biology.     

Proton-coupled bioenergetic processes in extremely alkaliphilic bacteria

Oxidative phosphorylation, which involves an exclusively proton-coupled ATP synthase, and pH homeostasis, which depends upon electrogenic antiport of cytoplasmic Na⁺ in exchange for H⁺, are the two known bioenergetic processes that require inward proton translocation in extremely alkaliphilic bacteria. Energy coupling to oxidative phosphorylation is particularly difficult to fit to a strictly chemiosmotic model because of the low bulk electrochemical proton gradient that follows from the maintenance of a cytoplasmic pH just above 8 during growth at pH 10.5 and higher. A large quantitative and variable discrepancy between the putative chemiosmotic driving force and the phosphorylation potential results. This is compounded by a nonequivalence between respiration-dependent bulk gradients and artificially imposed ones in energizing ATP synthesis, and by an apparent requirement for specific respiratory chain complexes that do not relate solely to their role in generation of bulk gradients. Special features of the synthase may contribute to the mode of energization, just as novel features of the Na⁺ cycle may relate to the extraordinary capacity of the extreme alkaliphiles to achieve pH homeostasis during growth at, or sudden shifts to, an external pH of 10.5 and above.     

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH₂QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH₂QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for ¹³C,¹⁵N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and ¹H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.     

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells

The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designated as 4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs). PAIB-SAs are active in the submicromolar to low micromolar range showing selectivity toward CYP1A1-expressing MCF7 cells as compared to cells devoid of CYP1A1 activity such as MDA-MB-231 and HaCaT cells. The most potent, PAIB-SA 13, bearing a trimethoxyphenyl group on ring B blocks the cell cycle progression in G2/M phase, disrupts the microtubule dynamics and is biotransformed by CYP1A1 into CEU-638, its potent antimicrotuble counterpart. Structure-activity relationships related to PAIB-SOs and PAIB-SAs evidenced that PAIB-SOs and PAIB-SAs are true bioisosteric equivalents fully and selectively activatable by CYP1A-expressing cells into potent antimitotics.