How the Krebs cycle disproves Darwinism (not!)

You know you're in for a treat when papers published in a (previously) reputable journal make frequent references to Dennis Noble and James Shapiro.

The purpose of this post is to demonstrate that you shouldn't let creationist amateurs publish anti-evolution rants in scientific journals.

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I want to discuss two papers that were recently published in the journal Progress in Biophysics and Molecular Biology. This used to be a very reputable journal but its reputation suffered a big blow in 2018 when it published a paper on panspermia. The current editor-in-chief at the time, Denis Noble, defended that article on the grounds that the origin of life is an unsolved problem and all points of view deserve to be covered in a scientific journal. Denis Noble is still on the editorial board along with Tom L. Blundell and Delphine Dean (see editorial board) and they now have to answer for publishing two creationist papers by Olen R. Brown and David A. Hullender.

Olen R. Brown is an emeritus professor from the University of Missouri (Columbus, Missouri, USA). His specialty is microbiology and he has written a book on miracles where he self-identifies as a Christian who claims that science should recognize God as creator (Miracles: Everything that Is or Was or Is to Come Is a Miracle). David A. Hullender is a professor of Mechanical and Aerospace Engineering at the University of Texas at Arlington (Texas, USA). They have collaborated on two papers that attack Darwinism.

Brown, O.R. and Hullender, D.A. (2022) Neo-Darwinism must Mutate to survive. Progress in Biophysics and Molecular Biology 172:24-38. [doi: 10.1016/j.pbiomolbio.2022.04.005]

Darwinian evolution is a nineteenth century descriptive concept that itself has evolved. Selection by survival of the fittest was a captivating idea. Microevolution was biologically and empirically verified by discovery of mutations. There has been limited progress to the modern synthesis. The central focus of this perspective is to provide evidence to document that selection based on survival of the fittest is insufficient for other than microevolution. Realistic probability calculations based on probabilities associated with microevolution are presented. However, macroevolution (required for all speciation events and the complexifications appearing in the Cambrian explosion) are shown to be probabilistically highly implausible (on the order of 10⁻⁵⁰) when based on selection by survival of the fittest. We conclude that macroevolution via survival of the fittest is not salvageable by arguments for random genetic drift and other proposed mechanisms. Evolutionary biology is relevant to cancer mechanisms with significance beyond academics. We challenge evolutionary biology to advance boldly beyond the inadequacies of the modern synthesis toward a unifying theory modeled after the Grand Unified Theory in physics. This should include the possibility of a fifth force in nature. Mathematics should be rigorously applied to current and future evolutionary empirical discoveries. We present justification that molecular biology and biochemistry must evolve to aeon (life) chemistry that acknowledges the uniqueness of enzymes for life. To evolve, biological evolution must face the known deficiencies, especially the limitations of the concept survival of the fittest, and seek solutions in Eigen's concept of self-organization, Schrödinger's negentropy, and novel approaches.

Brown, O.R. and Hullender, D.A. (2023) Biological evolution requires an emergent, self-organizing principle. Progress in Biophysics and Molecular Biology. [doi: 10.1016/j.pbiomolbio.2023.06.001]

In this perspective review, we assess fundamental flaws in Darwinian evolution, including its modern versions. Fixed mutations ‘explain’ microevolution but not macroevolution including speciation events and the origination of all the major body plans of the Cambrian explosion. Complex, multifactorial change is required for speciation events and inevitably requires self-organization beyond what is accomplished by known mechanisms. The assembly of ribosomes and ATP synthase are specific examples. We propose their origin is a model for what is unexplained in biological evolution. Probability of evolution is modeled in Section 9 and values are absurdly improbable. Speciation and higher taxonomic changes become exponentially less probable as the number of required, genetically-based events increase. Also, the power required of the proposed selection mechanism (survival of the fittest) is nil for any biological advance requiring multiple changes, because they regularly occur in multiple generations (different genomes) and would not be selectively conserved by the concept survival of the fittest (a concept ultimately centered on the individual). Thus, survival of the fittest cannot ‘explain’ the origin of the millions of current and extinct species. We also focus on the inadequacies of laboratory chemistry to explain the complex, required biological self-organization seen in cells. We propose that a ‘bioelectromagnetic’ field/principle emerges in living cells. Synthesis by self-organization of massive molecular complexes involves biochemical responses to this emergent field/principle. There are ramifications for philosophy, science, and religion. Physics and mathematics must be more strongly integrated with biology and integration should receive dedicated funding with special emphasis for medical applications; treatment of cancer and genetic diseases are examples.

These are remarkably juvenile papers. They read like the typical rants written by people who behave like adolescents on the creationist Facebook groups and blogs. They are mostly incoherent and they repeat practically all of the standard criticisms of evolution that we've been subjected to for the past 50 years plus some more modern attacks by the likes of Denis Noble and James Shapiro.

I want to concentrate on one of their main subjects; namely that the evolution of complex structures is so improbable that "survival of the fittest" is impossible. One of their examples in the Krebs cycle. They use it as a metaphor for speciation on the grounds that speciation requires an enormous number of improbable events, just like the Krebs cycle. Here's how they describe their calculations in the first paper.

We elected to calculate the probability (P) that mutations, in the genome of a living cell with no codes for any enzyme of the Krebs cycle, can create a genome containing all the unique enzymes required for a Krebs cycle which includes the four cytochromes essential for functional aerobic ATP (adenosine triphosphate) generation. ... We compare probabilities for creation of the functional Krebs cycle within one genome as required by the plain meaning of the statement of the selecting ability of survival of the fittest.

The authors are trying to prove that an irreducibly complex system like the Krebs cycle had to arise almost spontaneously because none of the individual components would work by themselves. They calculate the probability to be 10^-51. Assuming this is just one of the things required for speciation, this means that Darwinian survival of the fittest isn't an adequate explanation for speciation events—according to the authors.

If the overly simplified assumption is made that this speciation event must be repeated thousands or even millions of times in the process of evolution, then the probabilities of such a sequence of events is absurdly ridiculous; for example, if only 3 speciation events must occur in sequence, the probability of state #1 decreases to (10⁻⁵¹)³ = 10⁻¹⁵³.

From now on I'll refer to the Krebs cycle as the citric acid cycle. Let's see if Brown and Hullender understand enough biochemistry to be able to support such an extreme claim.

Here's a diagram of the citric acid cycle reactions shown as a unidirectional circular pathway in order to emphasize the breakdown of a 2-carbon unit (acetate, green) to produce 2 molecules of CO₂. This is somewhat misleading since all of the reactions are reversible (see below) but it emphasizes the point that the pathway is irreducibly complex. It will not work in the breakdown of acetate if you remove any one of the enzymes.

In order to understand the evolution of the citric acid cycle you need to understand some basic biochemistry. It's important to recognize that the citric acid cycle does not operate in isolation as a unidirectional CO₂-generating pathway. The intermediates in the pathway are connected to many other biosynthesis and catabolic pathways such as amino acid metabolism, fatty acid metabolism, and carbohydrate metabolism as shown in the figure below. This gives us a valuable clue about it's origin—it's main role in the beginning was in those other pathways.

The other thing we need to know is something that isn't taught very well in introductory biochemistry courses. Many enzymes have multiple different substrates. They often catalyze a reaction that depends only on the presence of certain functional groups. For example, an enzyme could catalyze a decarboxylation reaction to produce CO₂ from any molecule that had a -COOH group. This means that enzymes that are required in one pathway can catalyze reactions that might become important in another pathway. Over time there might be a gene duplication event and the enzymes for different pathways might diverge but still show traces of their common ancestry. But in the beginning a single enzyme would be sufficient to work in the original pathway and a newly evolving one.

We have a pretty good idea how the citric acid cycle pathway could have evolved from more simple non-cyclic pathways. I describe a possible scenario in my textbook (Moran et al., 2011 pp. 412-414 © Pearson/Prentice Hall) (see below). It may not be the exact way that the citric acid cycle evolved but that's not the point. The point is that we know for a fact that a complete citric acid cycle did not have to arise all at once because there are many species that have only parts of a complete cycle.

This is just one thing that any competent reviewer should have noticed in the two papers that were published. I can only conclude that the papers were not reviewed by competent reviewers and that the editors of the journal had ulterior motives for publishing such trash.

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13.9 Evolution of the Citric Acid Cycle

The reactions of the citric acid cycle were first discovered in mammals and many of the key enzymes were purified from liver extracts. As we have seen, the citric acid cycle can be viewed as the end stage of glycolysis because it results in the oxidation of acetyl CoA produced as one of the products of glycolysis. However, there are many organisms that do not encounter glucose as a major carbon source and the production of ATP equivalents via glycolysis and the citric acid cycle is not an important source of metabolic energy in such species.

We need to examine the function of the citric acid cycle enzymes in bacteria in order to understand their role in simple single-celled organisms. These roles might allow us to deduce the pathways that could have existed in the primitive cells that eventually gave rise to complex eukaryotes. Fortunately, the sequences of several hundred prokaryotic genomes are now available as a result of the huge technological advances in recombinant DNA technology and DNA sequencing methods.We can now examine the complete complement of metabolic enzymes in many diverse species of bacteria and ask whether they possess the pathways that we have discussed in this chapter. These analyses are greatly aided by developments in the fields of comparative genomics, molecular evolution, and bioinformatics.

Most species of bacteria do not have a complete citric acid cycle. The most common versions of an incomplete cycle include part of the left-hand side. This short linear pathway leads to production of succinate or succinyl CoA or α-ketoglutarate by a reductive process using oxaloacetate as a starting point. This reductive pathway is the reverse of the traditional cycle that functions in the mitochondria of eukaryotes. In addition, many species of bacteria also have enzymes from part of the right-hand side of the citric acid cycle, especially citrate synthase and aconitase. This allows them to synthesize citrate and isocitrate from oxaloacetate and acetyl CoA. The presence of a forked pathway (Figure 13.24) results in the synthesis of all the precursors of amino acids, porphyrins, and fatty acids.

There are hundreds of diverse species of bacteria that can survive and grow in the complete absence of oxygen. Some of these species are obligate anaerobes—for them, oxygen is a lethal poison! Others are facultative anaerobes—they can survive in oxygen free environments as well as oxygen-rich environments. E. coli is one example of a species that can survive in both types of environment. When growing anaerobically, E. coli uses a forked version of the pathway to produce the necessary metabolic precursors and avoid the accumulation of reducing equivalents that cannot be reoxidized by the oxygen requiring electron transport system. Bacteria such as E. coli can grow in environments where acetate is the only source of organic carbon. In this case, they employ the glyoxylate pathway to convert acetate to malate and oxaloacetate for glucose synthesis.

The first living cells arose in an oxygen-free environment over three billion years ago. These primitive cells undoubtedly possessed most of the enzymes that interconverted acetate, pyruvate, citrate, and oxaloacetate, since these enzymes are present in most modern bacteria. The development of the main branches of the forked pathway possibly began with the evolution of malate dehydrogenase from a duplication of the lactate dehydrogenase gene. Aconitase and isocitrate dehydrogenase evolved from enzymes that are used in the synthesis of leucine (isopropylmalate dehydratase and isopropylmalate dehydrogenase, respectively). (Note that the leucine biosynthesis pathway is more ubiquitous and more primitive than the citric acid cycle.)

Extension of the reductive branch continued with the evolution of fumarase from aspartase. Aspartase is a common bacterial enzyme that synthesizes fumarate from L-aspartate. L-aspartate, in turn, is synthesized by amination of oxaloacetate in a reaction catalyzed by aspartate transaminase (Section 17.3). It is likely that primitive cells used the pathway oxaloacetate → aspartate → fumarate to produce fumarate before the evolution of malate dehydrogenase and fumarase. The reduction of fumarate to succinate is catalyzed by fumarate reductase in many bacteria. The evolutionary origin of this complex enzyme is highly speculative but at least one of the subunits is related to another enzyme of amino acid metabolism. Succinate dehydrogenase, the enzyme that preferentially catalyzes the reverse reaction in the citric acid cycle, is likely to have evolved later on from fumarate reductase via a gene duplication event.

The synthesis of α-ketoglutarate can occur in either branch of the forked pathway. The reductive branch uses α-ketoglutarate:ferredoxin oxidoreductase, an enzyme found in many species of bacteria that don’t have a complete citric acid cycle. The reaction catalyzed by this enzyme is not readily reversible. With the evolution of α-ketoglutarate dehydrogenase the two forks can be joined to create a cyclic pathway. It is clear that α-ketoglutarate dehydrogenase and pyruvate dehydrogenase share a common ancestor and it is likely that this was the last enzyme to evolve.

Some bacteria have a complete citric acid cycle but it is used in the reductive direction to fix CO₂ in order to build more complex organic molecules. This could have been one of the selective pressures leading to a complete pathway. The cycle requires a terminal electron acceptor to oxidize NADH and QH₂ when it operates in the more normal oxidative direction seen in eukaryotes. Originally, this terminal electron acceptor was sulfur or various sulfates, and these reactions still occur in many anaerobic bacterial species. Oxygen levels began to rise about 2.5 billion years ago with the evolution of photosynthesis reactions in cyanobacteria. Some bacteria, notably proteobacteria, exploited the availability of oxygen when the membrane associated electron transport reactions evolved. One species of proteobacteria entered into a symbiotic relationship with a primitive eukaryotic cell about two billion years ago. This led to the evolution of mitochondria and the modern versions of the citric acid cycle and electron transport in eukaryotes. The evolution of the citric acid cycle pathway involved several of the pathway evolution mechanisms discussed in Chapter 10. There is evidence for gene duplication, pathway extension, retro-evolution, pathway reversal, and enzyme theft.

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Additional information on other enzymes in this pathway can be found in: On the Evolution of New Enzymes: Completely Different Enzymes Can Catalyze Similar Reactions and The evolution of citrate synthase.

For more information on succinate dehydrogenase and it's proper substrates and products see: Succinate Dehydrogenase; Succcinate Dehydrogenase and Evolution by Accident.

For more details on the relationship of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase see: Pyruvate Dehydrogenase Evolution.

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