Manfred Eigen: the realization of his vision of Biophysical Chemistry.

Manfred Eigen turned 90 on May 9th, 2017. He celebrated with a small group of colleagues and friends on behalf of the many inspired by him over his lifetime-whether scientists, artists, or philosophers. A small group of friends, because many-who by their breakthroughs have changed the face of science in different research areas-have already died. But it was a special day, devoted to the many genius facets of Manfred Eigen's oeuvre, and a day to highlight the way in which he continues to exude a great, vital and unbroken passion for science as well as an insatiable curiosity beyond his own scientific interests. He continues to dismiss arguments such as, that scientific problems cannot be solved because of a current lack of appropriate tools, or because of the persuasion of the community that certain things are immeasurable. He has lived up to and accepted only the highest scientific standards with his fundamental contributions in widely differing research fields, for which he has received numerous prizes and honorary doctorates, including the Nobel Prize for Chemistry in 1967. Some of his outstanding contributions to science and technology are honored in the following chapters. Here, we will report some characteristic traits of Manfred Eigen, and his personal development. We highlight his visionary foresight regarding how multidisciplinary science should combine to study the complex processes of life and its evolution in establishing an institute that applied biological, chemical, and physical methods, and how his vision became sustained reality.

The iron-sulphur protein RNase L inhibitor functions in translation termination.

The iron-sulphur (Fe-S)-containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co-immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35-21 or sup45-2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read-through defects in sup45-2. Interestingly, we find that although the Fe-S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe-S cluster mutant of RLI1 cannot suppress the read-through defects of sup45-2.

Mitochondria as we don't know them.

Biochemistry textbooks depict mitochondria as oxygen-dependent organelles, but many mitochondria can produce ATP without using any oxygen. In fact, several other types of mitochondria exist and they occur in highly diverse groups of eukaryotes - protists as well as metazoans - and possess an often overlooked diversity of pathways to deal with the electrons resulting from carbohydrate oxidation. These anaerobically functioning mitochondria produce ATP with the help of proton-pumping electron transport, but they do not need oxygen to do so. Recent advances in understanding of mitochondrial biochemistry provide many surprises and furthermore, give insights into the evolutionary history of ATP-producing organelles.

Euglena gracilis ribonucleotide reductase: the eukaryote class II enzyme and the possible antiquity of eukaryote B12 dependence.

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.

Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria.

Mitochondria perform a central function in the biogenesis of cellular iron-sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.

Early cell evolution, eukaryotes, anoxia, sulfide, oxygen, fungi first (?), and a tree of genomes revisited.

Genomes contain evidence for the history of life and furthermore contain evidence for lateral gene transfer, which was an important part of that history. The geological record also contains evidence for the history of life, and newer findings indicates that the Earth's oceans were largely anoxic and highly sulfidic up until about 0.6 billion years ago. Eukaryotes, which fossil data indicate to have been in existence for at least 1.5 billion years, must have therefore spent much of their evolutionary history in oxygen-poor and sulfide-rich environments. Many eukaryotes still inhabit such environments today. Among eukaryotes, organelles also contain evidence for the history of life and have preserved abundant traces of their anaerobic past in the form of energy metabolic pathways. New views of eukaryote phylogeny suggest that fungi may be among the earliest-branching eukaryotes. From the standpoint of the fungal feeding habit (osmotrophy rather than phagotrophy) and from the standpoint of the diversity in their ATP-producing pathways, a eukaryotic tree with fungi first would make sense. Because of lateral gene transfer and endosymbiosis, branches in the tree of genomes intermingle and occasionally fuse, but the overall contours of cell history nonetheless seem sketchable and roughly correlateable with geological time.

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions.

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.

A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes.

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.