An Interview with Manuel Varela and Ann Varela—About Albert Lehninger: Who Wrote Principles of Biochemistry?

May 28, 2020 by

Michael F. Shaughnessy

1) Those of us who graduated from college look back at our alma mater—but also, we tend to remember classic textbooks that got us through many courses. Albert Lehninger’s book Principles of Biochemistry helped us through perhaps one or two classes. But when and where was this productive scientist born, and what do we know about his early education?

Dr. Albert Lester Lehninger is known for his pioneering studies on the biochemical mechanism of oxidative phosphorylation. He is best known as the first author of the famous textbook Biochemistry: The Molecular Basis of Cell Structure and Function, studied by many an undergraduate or medical student for generations. Lehninger’s book is one of the most engaging and well-liked biochemistry textbooks of all time.

Lehninger was born ontheseventeen of February 1917, in Bridgeport, Connecticut, in the U.S. While we know little about Lehninger’s primary and secondary education, we do know that he took his B.A. degree in English from Wesleyan University in 1939. We also know that Lehninger obtained an M.S. in 1940 and Ph. D. in biochemistry in 1942 from the University of Wisconsin–Madison. From 1942 to 1945, Lehninger was an instructor at the University of Wisconsin–Madison. In 1945, he relocated to the University of Chicago, where he was a faculty member until 1952. During the same year, he moved to Johns Hopkins School of Medicine, where he assumed the title of DeLamar Professor while a member of the Department of Biological Chemistry.

2) We understand that he attended university. What were some of his accomplishments there?

His doctoral research encompassed the metabolism of acetoacetate and fatty acid oxidation by liver cells. In what may be considered his first publication and publishing as a single author, Lehninger reported his early studies in the Journal of Biological Chemistry, in March of 1942. He described his studies of acetoacetate breakdown into two molecules of acetic acid in rabbit muscle. He further noted an especially vigorous activity in the bacterium Escherichia coli under oxygen-free conditions. He suggested that an enzyme was responsible for the acetoacetate break up.

His graduate advisor at the time was Dr. Edgar J. Witzemann. The first journal article written together by Lehninger and Witzemann was submitted to the Journal of Biological Chemistry in late December of 1941 and published in April of 1942. The report presented a new laboratory method they had developed for the synthesis of acetopyruvic acid, known then also as α,γ-diketo-n-valeric acid. Witzemann and Lehninger further demonstrated that degradation of acetopyruvic acid resulted in the production of acetone and oxalic acid.

These studies in Professor Witzemann’s laboratory by Lehninger represent his first experience in research investigations. This research activity and the resulting data turned out to be quite relevant in metabolism. First, it addressed a connection between fatty acid oxidation and elements of intermediary metabolism. In later years, acetate would be involved in the form of acetyl coenzyme A and play critical roles in the oxidation of fatty acids when the Krebs cycle was saturated. Under conditions where the acetyl CoA is produced more than what the Krebs cycle can accommodate, then ketone bodies such as acetone, acetoacetate, and other compounds are made. Such a situation might occur in patients with diabetes.

3) In terms of research—was Lehninger a pure laboratory individual? Or were his contributions in terms of sharing knowledge or teaching?

A convincing argument can be made that Lehninger’s life and career encompassed elements of both laboratory work and teaching. First, his research investigations informed the necessary foundations of energetics and oxidative phosphorylation. From the efforts in the laboratory with Eugene Kennedy, they discovered that critical metabolic systems occurred in the matrix of the mitochondrion. During this same timeframe, between 1948 and 1951, Lehninger and Kennedy had also helped to demonstrate that several enzymes that played roles in performing biochemical reactions of glycolysis resided in the cytoplasm.

In 1951, Lehninger discovered that the insides of mitochondria undergo oxidation of NADPH. The term NADPH stands for nicotinamide adenine dinucleotide phosphate. As such, NADPH refers to sufficiently reduced, that is, harboring all possible electrons. On the other hand, NADP+ denotes that the molecule is oxidized, having lost electrons in the form of hydrides—atoms of hydrogen accompanied by electron particles. In Lehninger’s day, NADPH had been called DPNH, for diphosphopyridine nucleotide, and usually qualified as adequately reduced (DPNH) or oxidized (DPN). Lehninger further made an association between NADPH oxidation and the production of ATP, thus establishing that oxidative phosphorylation is coupled to electron transfer, with oxygen as the final acceptor of electrons in aerobic systems.

Lehninger first published Biochemistry in 1970. His rationale for writing the classic stemmed from his realization that the field of biochemistry had recently emerged from a subject that merely covered biological occurrences and enzymes that produced organic molecules. He noted, thus, that biochemistry could be described as a set of established principles. He viewed biochemistry as a molecular logic used by living organisms. He had blended biochemistry into four unifying subsets: biomolecules, energy production, energy utilization, and transfer of genetic information. Students from all walks of life loved it, and hundreds of such appreciative students wrote him heartening letters. The positive reviews moved him significantly.

His readership response motivated Lehninger to write an extensively revised and updated second edition, which he published in 1975. Every paragraph was amended, and new chapters were written. The second version had an updated historical timeline of biochemistry history. His original organizing principles were revised as follows: molecular components of cells, catabolism generating phosphate-bond energy, biosynthesis using the power of phosphate bonds, genetic information encompassing the Central Dogma of DNA replication, RNA synthesis, and translation.

After Lehninger’s death on the fourth of March in 1986 at the age of 69, authors Dave L. Nelson and Mike M. Cox continued with revising newer editions of the popular book first begun by Lehninger. Nelson received postdoctoral training under Eugene Kennedy, who, in turn, had been Lehninger’s first graduate student. As an undergraduate student, Cox reported that Lehninger’s first edition of Biochemistry was a significant influence and an inspiration to becoming a biochemist. Edition number two in 1993 was the last version with all three biochemists as co-authors. It is delightful to know that a careful reader can still see many elements of Albert Lehninger’s original edition nicely preserved in the latest publications. As a faculty member, one of us (MFV) gladly became an expert reviewer for several chapters of Nelson & Cox’s fifth edition of Lehninger Principles of Biochemistry. As of this writing, Nelson and Cox have published seven editions, the most recent one published in 2017.

Biochemistry, the study of chemical reactions inherent in the cells of all living beings, is critical for the life of all organisms. Failure in any aspect of the biochemical totality can result in severe medical disease. Biochemistry is also of tremendous utility to industry, biotechnology, bioinformatics, genomics, metagenomics, and molecular biology. There is scarcely doubt that the legacy of Lehninger will be his influence on the teaching of biochemistry to many new generations of students in most scientific fields of study.

4) He worked with Eugene Kennedy—and discerned that mitochondria are somehow linked with oxidative phosphorylation in eukaryotes. Can you explain this in layman’s language?

As briefly mentioned above, Eugene Kennedy was Lehninger’s first graduate student. Their work together would establish the biochemical pathways of fatty acid breakdown, the Krebs cycle, and that mitochondria are sites where oxidative phosphorylation takes place within eukaryotic cells. These discoveries marked the beginnings of the field of biological energetics, where biological systems undergo energy transduction using enzymes.

Figure – The Mitochondrion

Mitochondria are tiny membrane-enclosed packets that reside on the insides of most known eukaryotic cells. These intracellular packets, the mitochondria, are organelles that are commonly referred to powerhouses of the living cell. It is within these internal mitochondria that electrons are taken from oxidized nutrients consumed by living beings. All nutrients, whether fats, protein, carbohydrates, or nucleic acids, are used for energy production by shunting the extracted electrons to the respiratory chains that are located within the inner of the two mitochondrial membranes as an embedded series of proteins.

These embedded proteins constitute the respiratory chain, and they undergo a series of reduction (electron gain) and oxidation (electron loss) reactions. As these electron-transferring processes take place along the respiratory chain, protons (charged hydrogen atoms) are transported across the inner membrane from the matrix of the mitochondria to the space marked between the inner and outer membranes. These charged hydrogens collect there on one side of the inner membrane.

This unequal concentration of protons on one side of the membrane, i.e., in the intermembrane space and a correspondingly lower level of protons on the other side of this same inner mitochondrial membrane, form a new type of biological energy called the proton motive force or an electrochemical gradient of protons. This proton gradient is a natural living energy that results from the electron-transferring activity of the respiratory chain. As electrons are moved from protein to protein along the electron transport chain, protons are pumped out and form the proton motive force. Eventually, the electrons end up at oxygen, the final acceptor of the electrons that were extracted from food. Next, the proton motive force energy is used to phosphorylate ADP to produce ATP. Then, the ATP is used by its hydrolysis to help the living cell undergo its life processes.

This process described above is the basis of oxidative phosphorylation. The oxidative part is the electron transferring process—from nutrients to elements of the respiratory chain to oxygen. The phosphorylation aspect involves the addition of a phosphate to ADP, forming ATP. Thus, the oxidation is coupled directly to phosphorylation. Without this so-called “Ox-Phos,” there can be no life!

5) The modern study of energy transduction—what does this mean? Why is it significant, and what does Lehninger have to do with this?

Energy transduction is the conversion of one form of biological energy to produce another form of energy, called ATP. The starting energy can arrive at the living organisms in the form of sunlight or nutrients. In the case of photosynthesis, glucose and oxygen are converted to form carbon dioxide, water, and energy. In the case of non-photosynthetic chemotrophs, which harness power from chemicals produced by photosynthesis, pass along electrons to oxygen to form water, carbon dioxide, and other products by oxidizing the energy-rich molecules of plants. In the case of heterotrophs, which use products from autotrophs for nutrients, expel carbon dioxide back to the atmosphere. All three types of energy transducers invoke the flow of electrons to generate the electrochemical potential energy, which is afterward used to make ATP. All such electron carrying practices involve oxidation-reduction reactions.

Lehninger’s investigations established that oxidative phosphorylation occurred with the inner matrix of the mitochondrial organelles. Understanding where inside the cell that oxidative phosphorylation occurred was a significant discovery. Knowing its cellular location permitted investigators to explore energy transduction at the molecular level directly. Consequently, it allowed investigators to study the molecular aspect of all life, energy transduction. All other investigators of biochemistry interested in metabolism and energetics soon focused on the mitochondria as a resource for the study of the life-giving process of energy transduction.

We know in modern times, because of Lehninger’s pioneering work, that the inner membrane of the mitochondrion harbors the respiratory chain and site of the ATP-producing ATP synthase enzyme. We know that ATP synthase converts the energy of the electrochemical potential, generated by electron and proton flow, into a phosphorylating potential. We understand that the electrons flow from food to oxygen. We know that protons flow from the inside of the mitochondrion to the space between the inner and outer mitochondrial membranes to establish the bioenergetic driving force for phosphorylation. The result is the production of life-sustaining ATP.

6) Metabolism at a molecular level was linked to Lehninger—why is metabolism at the molecular level essential to understand?

One of Lehninger’s chief scientific contributions to biochemistry involves a deeper understanding of metabolism at the level of molecular systems. For example, his discoveries mentioned above established for the first time in scientific history that critically significant pathways of intermediary metabolisms, such as fatty acid oxidation and the Krebs cycle occur within a specific intracellular location, namely, the organelle called the mitochondrion. Furthermore, he discovered that the incredibly valued oxidative phosphorylation process occurs within the internal membrane of the mitochondria. In so doing, Lehninger helped to launch the imperative new field of bioenergetics.

File:2508 The Electron Transport Chain.jpg

Figure – The electron transport chain and ATP synthase

Today we know that because of these discoveries by Lehninger, succeeding generations of investigators have continued to acquire further knowledge about the molecular mechanisms of the players involved in bioenergetics. For instance, today, we know the molecular structures for all the components that constitute the electron transporting chains of the respiration process. We even know the various pathways of the electrons as they cycle through their respiratory proteins along the chain. Furthermore, we see the structures and enzymatic mechanisms that produce ATP as protons flow back into the ATP synthase molecule. We now understand how the electron carriers NADH and FADH2 shuttle the electrons that they extracted from nutrients and biochemicals of intermediary metabolism to complexes of the respiratory chain.

Lehninger’s work paved the way for Peter Mitchell to formulate his famous Nobel Prize-winning theory of chemiosmosis that the energy of the proton motive force was the driving force for ATP synthesis. Lehninger’s work made possible Paul Boyer’s elucidation of the molecular mechanism for ATP synthase, another Nobel worthy accomplishment.

7) What have I neglected to ask?

Lehninger was an unwitting and quite reluctant participant of the “Ox Phos Wars.” In the early 1960s, Dr. Peter Dennis Mitchell had formulated his Nobel-Prize idea of chemiosmosis. The chemiosmotic theory held that the bioenergy stored in proton motive force generated as electrons flowed through the respiratory chain was, in turn, used towards the biosynthesis of ATP. The Mitchell theory elegantly incorporated elements of Lehninger’s oxidative phosphorylation ideas into the chemiosmotic mechanism, and Lehninger was supportive of the hypothesis in general.

Unfortunately, at the time, many investigators in the bioenergetics field did not agree at all with Mitchell’s postulated role for the proton motive force and its coupling of oxidation via electron transport to ATP synthesis. The controversy was the essence of the Ox Phos Wars—one group was pro-Mitchell and, thus, pro-chemiosmotic theory, and the other group was emphatically anti-chemiosmosis.

In the mid-1960s, Lehninger inadvertently became a casualty of the Ox Phos Wars when his laboratory collected stoichiometric data regarding the number of protons that were extruded as a function of oxygen atoms during electron transport. His data predicted that as the electrons transferred from complex to complex along the respiratory chain to oxygen (the final electron acceptor), four protons were extruded per complex along the chain. The Lehninger data meant that if the three proton-translocating complexes along the chain each ejected four protons, then 12 protons were translocated per oxygen atom. While the Lehninger data did not argue against Mitchell’s chemiosmotic theory, Mitchell, nevertheless, believed that it did! Lehninger had thought that Mitchell’s theory merely needed revision, not abandonment.

Conversely, Mitchell’s theory had already mathematically worked out the chemical stoichiometry. Mitchell postulated one ATP molecule was produced per two protons transported through each of the three complexes as two electrons were passed through the chain, predicting six protons per oxygen atom. Lehninger’s data predicted 12 protons per oxygen atom. This difference in the number of protons transported per oxygen atom was the essence of the disagreement between Lehninger and Mitchell.

Mitchell and Lehninger tried to sort it out by correspondence, but the conversations got increasingly confrontational. Mitchell argued that Lehninger misinterpreted his own data. Irrationally, Mitchell felt any adjustment in proton transport numbers meant the entire chemiosmotic theory collapsed.

Mitchell had grown accustomed to encountering vigorous attacks on his theory, and any criticisms, however subtle, would be viewed as a strike on the entire notion of chemiosmosis. Mitchell placed Lehninger’s disagreement on proton number per oxygen atom directly into this adversarial category.

The controversy over the proton per oxygen math did not get resolved for almost 15 years! In the interim, two groups had emerged: 12 protons (the Lehninger camp) versus the 6 protons (the Mitchell camp). The case for Lehninger’s 12-proton adjustment grew with time as new data for it accumulated. Experimental refinements by many laboratories in the proton number ended up with 10 protons translocated per oxygen atom: four protons extruded through complex I; four protons per complex III, and two protons per complex IV.

Mitchell had eventually thrown in the towel and acquiesced. He had been wrong about stoichiometry. He was also wrong about the notion that changes or refinements would derail chemiosmosis. In the end, chemiosmotic theory had survived the proton number adjustment after all. Mitchell took the Nobel in chemistry in 1977.

As one views the controversy with hindsight, one can appreciate the great diversity of the respiratory chains harbored in the potentially billions if not trillions of species on Earth and their corresponding level of diversity in their respiratory chain stoichiometries! Surely, respiratory chains elucidated in the future will support one’s preferred proton-oxygen stoichiometry!

Lehninger once was quoted as saying, Virtually every chemical reaction in a cell occurs at a significant rate only because of the presence of enzymes.” Surprisingly, Lehninger did not get the Nobel. He was, however, widely recognized by other awarding vehicles. For instance, he was the proud recipient of the Pfizer Award in Enzyme Chemistry (1948), a Guggenheim Fellowship (1951), Election to the National academy of Sciences (1956), the Remsen Award of the American Chemical Society and the coveted Passano Foundation Award.

In addition to writing Biochemistry, an endearing textbook for undergraduates, graduates, and students of professional schools, he wrote several notable works devoted to cellular structure and behavior: The Mitochondrion, and Bioenergetics.

Readers with interest in the life and science of Lehninger are encouraged to visit these sites:

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