Redox Pioneer: Professor Leopold Flohé

Glutathione peroxidases as selenoenzymes and as the prototypes of the non-heme peroxidases

Studies on the catalytic mechanism and the peculiar kinetics of GPx1, carried out by Leopold Flohé, preceded the discoveries of the other glutathione peroxidases (GPxs) and of peroxiredoxins (Prxs) that constitute the present scenario of a network of oxidoreductases controlling redox transitions involved in both antioxidant defense and signaling. The discovery that GPx1 contains selenium and that the element is inserted into redox-active proteins as selenocysteine, boosted the innovative field of biochemical selenology, which expanded from the low-molecular-weight compounds contained in plants to bacterial and animal selenoproteins (1). How the early achievements in selenology developed into a now flourishing field of research is the subject of a recent essay written by Leopold Flohé himself (8). Further, the discovery of glutathione peroxidase homologues containing sulfur instead of selenium (CysGPxs) opens new evolutionary horizons. The alternative use of these elements brings into focus the biologic advantage of having selenium rather than sulfur in some of these enzymes (32). Another intriguing aspect of CysGPxs is the switch from glutathione peroxidase to thioredoxin peroxidase activity, which was first described for a GPx of Plasmodium falciparum by Dr. Flohé (43) and has increased interest as an important means of thioredoxin-mediated redox regulation.

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Leopold Flohé with (from right) Regina Brigelius-Flohé, Matilde Maiorino, and Fulvio Ursini at the SFRR Meeting 2006 in Davos

The creative atmosphere of the “university village” Tübingen, which inspired the young Otto Warburg, a founder of redox biochemistry, as well as other giants of biochemistry, such as Felix Hoppe-Seyler, Friedrich Miescher, and Adolf Butenandt, must have primed the student Leopold Flohé. In his MD thesis, he had disappointed his supervisor with negative results on the supposedly different redox potentials of fetal versus adult hemoglobin and decided to dig deeper into the physiology of red blood cells to determine why red blood cells from newborns turn brown more rapidly than do those of adults. He therefore persuaded his biochemistry supervisor to let him begin a diploma thesis on “Glutathion-Peroxidase,” an enzyme described by Gordon Mills in 1957 (35), but, as Poldi learned much later, was declared not to exist at all at a FASEB Meeting in the United States. Apparently only heme peroxidases, which display an unequivocal spectrum, were accepted as catalyzing hydroperoxide processing. Having purified GPx from an enormous amount of bovine blood, Leopold Flohé produced a series of rigorous studies on the enzymology and kinetics of GPx1 (5, 16). Thereby he demonstrated that the enzyme contained neither heme, nor flavin, nor any other known coenzyme but was nevertheless superior to the heme peroxidases in terms of kinetic parameters and specificity. The dogma that hydroperoxides can be processed only by heme-containing enzymes finally had to be revised.

Glutathione peroxidase as a selenoenzyme

In 1972, an abstract of the Federation proceedings from Hoekstra's group in Wisconsin reported that, in a model of rat Se deficiency, GPx activity was low, and radioactive selenium, given to Se-deficient rats, co-eluted with GPx1 (39). This observation prompted Leopold Flohé to search for the element in the last tiny aliquot of purified GPx1 left over from kinetic studies. He took advantage of a γ-spectrometer made available to German geologists for analyzing stones brought back from the moon by the Apollo mission. The measurements clearly yielded a precise stoichiometry of four atoms of selenium for one molecule of the tetrameric enzyme, verifying the selenoprotein nature of GPx1 (14). Thus, the moon had its second affair with chemistry, after having lent its Greek name Σεληνη to the element when it was discovered in 1817 by Berzelius.

The connection between selenium and metabolic processes, as established by the groups of Flohé and Hoekstra, had important practical consequences: it enforced a revision of the appreciation of selenium and enabled the FDA to approve Se supplementation for domestic animals in 1979 and for humans in 1981 (36).

During his industrial career at the Grünenthal GmbH in Aachen, Leopold Flohé resumed his GPx research. With Wolfgang A. Günzler and Gert Steffens, he established the first full-length sequence of a selenoprotein, which unequivocally demonstrated that its selenium moiety, selenocysteine, was inserted in the peptide chain as the 21^st naturally occurring amino acid (22). This finding was instrumental in the identification of TGA as the universal selenocysteine codon (3, 48).

From tetrameric GPx1 to monomeric GPx4

We first met Leopold Flohé in the early 1980s at a Congress in Munich, mumbling in front of our poster, which reported on a monomeric GPx with an antioxidant activity on membranes. His concern was that our monomeric peroxidase could just be a monomeric variant of GPx1. The impossibility of solving the problem just by discussing it, impelled us to investigate. Finally, Poldi's team in Aachen, by partial amino acid and full genomic DNA sequencing, proved that our peroxidase was indeed a distinct protein, now called GPx4 (2, 40). A second mammalian selenoenzyme had come to light.

Poldi's curiosity-driven persistence to answer the question of what the novel gene product might be really good for continued to be fruitful. It led to the paradigm of functional diversity among homologous enzymes that share both active-site and catalytic mechanisms (47). In this context, a major achievement of our ongoing scientific collaboration is the discovery of a functional role of GPx4 that is just the opposite of an antioxidant defense. We demonstrated that, in the final phase of spermatogenesis, GPx4 catalyzes the oxidative protein thiol cross-linking, thereby moonlighting (changing function) into a structural protein of spermatozoa (46). This novel physiologic function not only unraveled the previously enigmatic role of selenium in male fertility; it can also be seen as the prototype of a posttranslational protein modification relevant to cell signaling (10).

Hydroperoxide-processing enzymes in pathogens

In 1995, Leopold Flohé decided to leave all of his administrative jobs and to enjoy science full-time. He concentrated his efforts on a multifaceted project of “oxidative stress” that differed from the most fashionable analysis of the defense against ROS in humans. Instead, he pioneered a series of studies on antioxidant defense in pathogens, aiming to find molecular targets for designing new drugs to fight major diseases, such as trypanosomiasis, leishmaniasis, and tuberculosis, which affect the most neglected populations of the planet (15, 37, 45).

Within an amazingly short time, he with his new and young team clarified the hydroperoxide metabolism in trypanosomatids with the identification of two novel enzymes, tryparedoxin and tryparedoxin peroxidase (37). The former proved to be a remote relative of thioredoxin; the latter was homologous to the “thiol-dependent antioxidant protein” of yeast, which had been discovered by Earl Stadtman's team (29) and later was characterized as a thioredoxin peroxidase. These two new peroxidases, together with alkylhydroperoxide reductases (44) and a number of functionally poorly characterized “factors,” constituted the youngest peroxidase family, now known as peroxiredoxins (18).

The next challenge was the hydroperoxide metabolism in mycobacteria, which are devoid of GSH and a functional AhpF/AhpC system. Again, antioxidant defense in these organisms was proven to be dominated by redoxin-fueled peroxiredoxins (27).

With these studies, Leopold Flohé provided the basis for the identification, in-depth characterization, and validation of attractive molecular targets, which are now being exploited for drug design at the Molisa GmbH and elsewhere (26, 28).

Mitochondria as a source of superoxide

The finding of selenium in GPx was achieved a few years after the identification by McCord and Fridovich (33) of the redox activity of another protein of red blood cells containing copper and zinc, erythrocuprein, now known as superoxide dismutase, SOD1. These discoveries brought attention to the fundamental features of the integrated enzymatic system controlling the concentration of “free radicals” and other “reactive oxygen species.”

In this field, Dr. Flohé, in collaboration with his students Garriet Loschen and Christoph Richter and the colleagues Angelo Azzi and Britton Chance, authorized classic articles showing for the first time that mitochondria are an important source of superoxide (30, 31). These early publications remained unacknowledged for some time, but have more recently been rediscovered (e.g., in the context of TNF-α-triggered apoptosis (19, 25, 38). Related methodologic articles for the accurate determination of specific activities of SOD and GPx became quotation classics (13, 17).

Beyond “redoxology”, a scientific pioneer in transferring science to application

As evident from his employment over the years, Leopold Flohé must also have been a successful manager. While at Grünenthal GmbH, he developed biotechnologic procedures for the production of medications heading toward an integrated treatment of vascular occlusions and reperfusion injury with fibrinolytics (6), SOD (7), and prostacyclin analogues (9). Under his coordination, urokinase/pro-urokinase evolved through the long pathway from basic research (23, 42) to preclinical (4, 21) and clinical development (34) and production in an industrial scale (20).

Also at Grünenthal, he developed Tramadol, a best-selling analgesic (12). Finally, he became intimately involved in the elucidation of the teratogenic (11, 24) and neurotoxic (41) side effects of thalidomide, which had remained a persistent burden of the Grünenthal company.

While tasting a glass of wine and referring to his scientific adventures (8), we remember Poldi saying, “I am not the type who falls in love with a molecule just because it has an odd number of electrons, or gets excited about a method that promises the glamour of creativity and novelty. My driving force simply was to make something useful out of available knowledge and technology, and open eyes and serendipities often helped to pick a productive way to success.” This further corroborated our way of viewing him as an illuminist with a romantic trait.

After retirement, Dr. Flohé founded MOLISA GmbH, an innovative company that offers chemical and biologic high-tech services for the discovery and development of novel therapeutics. Since 2008, he has taught as Guest Professor at the Otto-von-Guericke-Universität, Magdeburg, Germany, and tries to coordinate European activities aiming at new therapeutic strategies against protozoal diseases as Chair of the COST Action CM0801, “New Drugs for Neglected Diseases.”