1. Introduction

The concept of proton transport chains (PTCs) was deduced from the substrate-channelling hypothesis, which was experimentally demonstrated by Srivastava and Bernhard [14–16]. Critics of the substrate-channelling hypothesis have argued that free diffusion minimizes any effects possibly provoked by substrate channelling [17]. We do not agree with this criticism for the following reasons. First of all, the substrate discussed to be transferred between two dehydrogenases is NADH [16]. Instead, we assert that the energy-rich NADH-H⁺ is product/substrate of the dehydrogenases. If free diffusion of the acid NADH-H⁺ into the cytosol occurs, as an acid, NADH-H⁺ will react with water to yield H⁺[H₂O]_n. Consequently, free diffusion of NADH-H⁺ entails the change from an active acid to an inactive salt. During reduction processes, it is clear that NADH-H⁺ transfers two H. Therefore, a rationale based on free diffusion would lead to inactivation of the reducing capacity of the co-enzyme NADH-H⁺. Second, the binding affinity of dehydrogenases for the co-enzyme excludes a concept based on free diffusion [18]. The binding of NADH-H⁺ to an enzyme frees the co-enzyme from the hydration layer. It is exactly this water-free binding of substrates to enzymes that allowed us to develop the PTC hypothesis. The term [mol/L] only applies to dissolved substrates. When bound to an enzyme, a substrate is no longer part of the aqueous layer, and both the position and movement of the substrate are defined, not random. This precise positioning stabilizes a specific substrate conformation, one prerequisite for optimal enzymatic activity.

2. The Proton Transport Chain Hypothesis

Our PTC hypothesis ensures that a water-free, intra-complex transfer of NADH-H⁺ maintains the activity of the co-enzyme. The PTC hypothesis completely changes the overriding perspective of biological processes based on emitting entropy to a model of producing entropy. In addition, the changes include the mathematical models used to calculate enzyme kinetics. For example, water-free conditions (mathematically) entail an infinite concentration [mol/L] of the substrate and thereby exclude the application of a great number of commonly used mathematical formulae used to calculate enzyme kinetics. An infinite concentration changes enzyme kinetics from concentration dependency to complex kinetics and provision of substrate (mol/s).

In vivo, enzymes such as muscle lactate dehydrogenase (LDH-m) and LDH-h act unidirectionally [19]. However, when they are investigated as isolated enzymes in vitro, they are disconnected from the flow of energy and material and act in a reversible manner. The PTC hypothesis provides a mechanism explaining the observations in vivo that are not reproducible using traditional methods in the laboratory. The PTC hypothesis also integrates the well-known fact that in vivo, enzymes exist as organized complexes. Thus, we assert that the water-free NADH-H⁺ transfer from the proton donor, glyceraldehyde 3-phophate dehydrogenase (GAPDH) to the proton acceptor, LDH-m unidirectionally drives the reduction of pyr⁻ to lac⁻. Thus, in vivo, LDH-m (in complex with GAPDH) only catalyses in the opposite direction the name of the enzyme suggests [20,21]. Citrate synthase is traditionally thought to be an enzyme that catalyses in only one direction. However, a couple of recent studies have reported citrate synthase enzymes capable of catalysing reversibly [22,23]. The enzymes characterized are both from bacteria, namely a sulphur-reducing anaerobic deltaproteobacterium, Desulfurella acetivorans [22], and a chemolithotrophic bacteria, Thermosulfidibacter takaii ABI70S6^T [23]. As bacteria do not have mitochondria, it is extremely difficult to draw comparisons with what happens in a eukaryotic cell. Metabolic enzymes within a eukaryotic cell are compartmentalized, separated by multiple membranes, exposed to different pH levels and are known to exist in enzyme complexes. T. takaii ABI70S6^T acquires the carbon for metabolic processes from CO₂ in their environment. Therefore, considering that eukaryotes generate CO₂ from glucose acquired from their environment, it is not surprising that these bacteria possess a citrate synthase capable of driving a reverse TCA cycle. Interestingly, characterization of the enzymes was performed in vitro using recombinant enzymes. Moreover, anaerobic respiration was performed via fumarate reductase and not via succinate dehydrogenase (SDH)/complex II of the Citric Acid Cycle. Finally, we do not believe that the discovery that bacterial citrate synthase enzymes act reversibly in vitro has any bearing on the eukaryotic Citric Acid Cycle concept. First, the eukaryotic enzyme is present on the human chromosome 12q13.3 and not a product of the mitochondrial genome. Therefore, it has likely diverged from its bacterial ancestor. Second, the condensation reaction catalysed by eukaryotic citrate synthase is practically irreversible, as it has a ΔG⁰′ of −7.7 kcal/mol (−32.2 kJ/mol) [24].

Combining enzyme complexes with PTCs creates a new and completely different understanding in Biology. The well-established didactically based sorting of glycolytic enzymes suggests single enzymes perform glycolysis, whereas our concept organizes enzymes into complexes optimizing energy and material transfer. A critical step in applying the tentative Fourth Law of Thermodynamics to biological processes is the identification of the nature of the energy entity, ordering organisms. Glucose metabolism permanently creates the energy entity H⁺. The energy of a H⁺ is high. The absolute hydration free energy of the proton, ΔG_hyd(H⁺), has been quoted in the literature to be from −252.6 kcal/mol to −262.5 kcal/mol, which corresponds to approximately 35-times the energy released by the hydrolysis of 1 mol of ATP [25]. Thus, H⁺ is always solvated, usually disolvated [26], and ‘free’ protons only exist in a chemical reaction written on a piece of paper. We illustrate this by combining H⁺ with proton carriers, such as H⁺[H₂O]_n, NADH-H⁺, lactic acid (lacH), pyrH and H₂CO₃.

This entry is adapted from the peer-reviewed paper 10.3390/ijms22020604