Difference between revisions of "Category:Ambiguity crisis - NAD and H+"

Hydrogen ion ambiguities in the electron transfer system

Communicated by Gnaiger E (2023-10-08) last update 2023-11-10

Electron (e^-) transfer linked to hydrogen ion (hydron; H⁺) transfer is a fundamental concept in the field of bioenergetics, critical for understanding redox-coupled energy transformations.

However, the current literature contains inconsistencies regarding H⁺ formation on the negative side of bioenergetic membranes, such as the matrix side of the mitochondrial inner membrane, when NADH is oxidized during oxidative phosphorylation (OXPHOS). Ambiguities arise when examining the oxidation of NADH by respiratory Complex I or succinate by Complex II.

Oxidation of NADH or succinate involves a two-electron transfer of 2{H⁺+e^-} to FMN or FAD, respectively. Figures indicating a single electron e^- transferred from NADH or succinate lack accuracy.

The oxidized NAD⁺ is distinguished from NAD indicating nicotinamide adenine dinucleotide independent of oxidation state.

NADH + H⁺ → NAD⁺ +2{H⁺+e^-} is the oxidation half-reaction in this H⁺-linked electron transfer represented as 2{H⁺+e^-} (Gnaiger 2023). Putative H⁺ formation shown as NADH → NAD⁺ + H⁺ conflicts with chemiosmotic coupling stoichiometries between H⁺ translocation across the coupling membrane and electron transfer to oxygen. Ensuring clarity in this complex field is imperative to tackle the apparent ambiguity crisis and prevent confusion, particularly in light of the increasing number of interdisciplinary publications on bioenergetics concerning diagnostic and clinical applications of OXPHOS analysis.

Supplement 1. The CI substrate is NADH + H⁺

In mitochondrial preparations, NADH-generating substrates, N, of various dehydrogenases (pyruvate, glutamate, malate, or other ET-pathway competent N-type substrate combinations) are applied to support respiration through Complex I (Gnaiger 2020). Importantly, malate and other N-type substrates are not CI-substrates.

xx Bottje WG (2019) Oxidative metabolism and efficiency: the delicate balancing act of mitochondria. Poult Sci 98:4223-30. - »Bioblast link«

Fig. 1 of Bottje (2019): NADH+H⁺ should be indicated as substrate of CI compared to succinate as substrate of CII. Their oxidation is a 2-electron reaction.

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Supplement 2. Electron transfer from CI ⟶ CII ⟶ CIII

The term electron transfer system takes into account the convergent structure of mitochondrial pathways merging from various branched routes at the N-junction and Q-junction (Gnaiger 2020). Unfortunately the term electron transport chain is still used in some branches of the bioenergetic literature. Combined with respiratory Complexes defined in numerical sequence as CI, CII, CIII, and CIV, this led to misrepresenting electron transfer as (NADH, FADH₂) → CI → CII → CIII:

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Supplement 3. NADH + H⁺ ⟶ NAD⁺ + (2)H⁺ (+2e^-)

It is important to distinguish in the oxidation of NADH the H⁺ that is consumed as a substrate (on the left side of the equation) from the 2H⁺ that are donated in the redox reaction of H⁺-linked electron transfer to the reductant FMN:

NADH + H⁺ ⟶ NAD⁺ + 2{H⁺+e^-}

In H⁺-linked electron transfer, the 2e^- should be matched with 2H⁺.

xx Mathur D, Riffo-Campos AL, Castillo J, Haines JD, Vidaurre OG, Zhang F, Coret-Ferrer F, Casaccia P, Casanova B, Lopez-Rodas G (2017) Bioenergetic failure in rat oligodendrocyte progenitor cells treated with cerebrospinal fluid derived from multiple sclerosis poatients. Front Cell Neurosci 11:209. - »Bioblast link«

Fig. 5 of Mathur et al (2017): H⁺ is consumed in the redox chemical (scalar) reaction of H⁺-linked electron transfer catalyzed by CI, NADH + H⁺ → NAD⁺ + 2H⁺. This same H⁺ is shown to be transported in the vectorial H⁺ translocation from the matrix side across the mtIM (H⁺_neg → H⁺_pos). The scalar and vectorial transformations must be distinguished. When the path is shown of the single electron (instead of 2{H⁺+e^-}) from NADH to ubiquinone, then it is particularly confusing when the 2H⁺ in the H⁺-linked electron transfer are indicated as remaining in the matrix.

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xx Rosca MG, Vazquez EJ, Chen Q, Kerner J, Kern TS, Hoppel CL (2012) Oxidation of fatty acids is the source of increased mitochondrial reactive oxygen species production in kidney cortical tubules in early diabetes. Diabetes 61:2074-83. - »Bioblast link«

Fig. 5 of Rosca et al (2012): Oxidation of succinate to fumarate reduces ubiquinone to ubiquinol through oxidation of FAD to FADH₂ and further redox steps in CII. Likewise, oxidation of NADH + H⁺ to NAD⁺ reduces ubiquinone to ubiquinol through oxidation of FMN to FMNH₂ and further redox steps in CI. This reduction of UQ to UQH₂ effectively consumes the 2H⁺ together with 2e^-. Hence, the 2H⁺ should not be shown to be formed in the matrix. Indication of H⁺-linked electron transfer as 2{H⁺+e^-} eliminates the ambiguity.

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## Nakane M (2020) Biological effects of the oxygen molecule in critically ill patients. J Intensive Care 8:95. - »Bioblast link«

## Copied with permission from: Hall J (2016) Guyton and Hall Textbook of Medical Physiology. 13. Elsevier, Philadelphia.

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Supplement 4. NADH ⟶ NAD⁺ + H⁺ (+ 2e^-)

In the chemical equation for oxidation of NADH + H⁺ by CI,

NADH + H⁺ → NAD⁺ + 2{H⁺+e^-}

H⁺ linked to electron transfer is indicated as 2{H⁺+e^-} on the product side, in contrast to H⁺ defined as a substrate in the chemical reaction (Gnaiger 2023). This formal distinction avoids an ambiguity arising from writing this equation as (see above),

NADH + H⁺ → NAD⁺ + 2H⁺

Then H⁺ on both sides of the equation may be considered to cancel, which provides a possible explanation for the frequent occurrence of the erroneous rearrangement as

NADH → NAD⁺ + H⁺

4.1. NADH ⟶ NAD⁺ + H⁺ + 2e^-

Electron flow from NADH to FMN in CI proceeds as a 2e^- transfer. The 2e^- are never free floating in the matrix but belong to the arrow reaching ubiquinone UQ. 2-electron transfer is linked to 2H⁺ transfer, hence the meaning of the single H⁺ — written on the right side of the equation — is elusive.

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4.2. NADH ⟶ NAD⁺ + H⁺ + e^-

The 2-electron transfer from NADH to CI is not well depicted in graphical representations suggesting the equation NADH ⟶ NAD⁺ + H⁺ + e^-.

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Fig. 1 by Głombik et al (2021): Red arrows indicate e^- transfer from NADH through CI to Q and from FADH₂ through CII to Q. In oxidation of NADH, one blue H⁺ appears in the matrix, which is pumped across the mtIM through CI. In fact, reducing equivalents are not pumped but are consumed in the reduction of FMN and further of UQ to UQH₂. 2H⁺ appear in the matrix in oxidation of FADH₂. Their fate is not clear (CII is not a H⁺ pump).

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4.3. NADH ⟶ NAD⁺ + H⁺

In many cases the oxidation NADH ⟶ NAD⁺ + H⁺ by CI is compared with oxidation by CII of succinate to fumarate, succinate to fumarate + 2H⁺, FADH₂ to FAD, or FADH₂ to FAD + 2H⁺. These combinations underscore the ambiguous nature in these portrayals of the transfer of reducing equivalents. What is the meaning of H⁺ in the equation NADH ⟶ NAD⁺ + H⁺ suggested by the following graphical representations of NADH oxidation by CI?

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Fig. 1 by Bejali et al (2020): The meaning of e^- attached to various enzymes is not clear. For indication of electron transfer, corresponding arrows should be added.

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Labelling FAD as 'Free FAD' is incorrect, since the prosthetic group FAD/FADH₂ remains covalently bound to the subunit SDHA of CII in the catalytic cycle.

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Fig. 9.1 Arrows are missing from substrates to products, which are required to make this graph meaningful.

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Supplement 5. NADH ⟶ NAD + H⁺ (+ e^-)

NAD is the IUPAC symbol for nicotinamide adenine dinucleotide without implication of its oxidation state, whereas the oxidized form of NAD is NAD⁺ and the reduced form of NAD is NADH (in terms of total amount, NAD = NADH + NAD⁺).

5.1. NADH ⟶ NAD + H⁺ + e^-

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5.2. NADH ⟶ NAD + H⁺

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Supplement 6. NADH ⟶ NAD⁺ + 2H⁺ (+ 2e^-)

A bit of bioenergetic mystery is the origin of the form of the chemical reaction supposedly catalyzed by CI,

NADH → NAD⁺ + 2H⁺

If H⁺-linked electron transfer would be indicated, then the 2H⁺ should be directed together with 2e^- to the prosthetic group FMN bound to CI instead of being pushed into the matrix space, and a H⁺ is missing on the substrate side. The unambiguous form of the equation is (Gnaiger 2023),

NADH + H⁺ → NAD⁺ + 2{H⁺+e^-}

6.1. NADH ⟶ NAD⁺ + 2H⁺ + 2e^-

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xx Yin M, O'Neill LAJ (2021) The role of the electron transport chain in immunity. FASEB J 35:e21974. - »Bioblast link«

6.2. NADH ⟶ NAD⁺ + 2H⁺ + e^-

xx Gallinat A, Vilahur G, Padró T, Badimon L (2022) Network-assisted systems biology analysis of the mitochondrial proteome in a pre-clinical model of ischemia, revascularization and post-conditioning. Int J Mol Sci 23:2087. - »Bioblast link«

S6.2

## Ignatieva E, Smolina N, Kostareva A, Dmitrieva R (2021) Skeletal muscle mitochondria dysfunction in genetic neuromuscular disorders with cardiac phenotype. Int J Mol Sci 22:7349. - »Bioblast link«

S6.2

6.3. NADH ⟶ NAD⁺ + 2H⁺

## Dilliraj LN, Schiuma G, Lara D, Strazzabosco G, Clement J, Giovannini P, Trapella C, Narducci M, Rizzo R (2022) The evolution of ketosis: potential impact on clinical conditions. Nutrients 14:3613. - »Bioblast link«

S6.3

xx El-Gammal Z, Nasr MA, Elmehrath AO, Salah RA, Saad SM, El-Badri N (2022) Regulation of mitochondrial temperature in health and disease. Pflugers Arch 474:1043-51. - »Bioblast link«

S6.3

xx Yu-Wai-Man P, Griffiths PG, Chinnery PF (2011) Mitochondrial optic neuropathies - disease mechanisms and therapeutic strategies. Prog Retin Eye Res 30:81-114. - »Bioblast link«

S6.3

Supplement 7. NADH ⟶ NAD + (2H⁺) + (2e^-)

NAD is the IUPAC symbol for nicotinamide adenine dinucleotide without implication of its oxidation state, whereas the oxidized form of NAD is NAD⁺ and the reduced form of NAD is NADH (in terms of total amount, NAD = NADH + NAD⁺).

7.1. NADH ⟶ NAD + 2H⁺ + 2e^-

xx Kataoka N, Matsutani M, Matsushita K (2020) Respiratory chain and energy metabolism of Corynebacterium glutamicum. In: Inui M, Toyoda K (eds) Corynebacterium glutamicum. Microbiology Monographs 23. Springer, Cham. - »Bioblast link«

S7.1

xx Shields HJ, Traa A, Van Raamsdonk JM (2021) Beneficial and detrimental effects of reactive oxygen species on lifespan: a comprehensive review of comparative and experimental studies. Front Cell Dev Biol 9:628157. - »Bioblast link«

S7.1

xx Wu Z, Ho WS, Lu R (2022) Targeting mitochondrial oxidative phosphorylation in glioblastoma therapy. Neuromolecular Med 24:18-22. - »Bioblast link«

S7.1

7.2. NADH ⟶ NAD + 2e^-

xx Yang J, Guo Q, Feng X, Liu Y, Zhou Y (2022) Mitochondrial dysfunction in cardiovascular diseases: potential targets for treatment. Front Cell Dev Biol 10:841523. - »Bioblast link«

S7.2

7.3. NADH ⟶ NAD + e^-

xx Chi SC, Cheng HC, Wang AG (2022) Leber hereditary optic neuropathy: molecular pathophysiology and updates on gene therapy. Biomedicines 10:1930. - »Bioblast link«

S7.3

xx Geng Y, Hu Y, Zhang F, Tuo Y, Ge R, Bai Z (2023) Mitochondria in hypoxic pulmonary hypertension, roles and the potential targets. Front Physiol 14:1239643. - »Bioblast link«

S7.3

xx Simon L, Molina PE (2022) Cellular bioenergetics: experimental evidence for alcohol-induced adaptations. Function (Oxf) 3:zqac039. - »Bioblast link«

S7.3

7.4. NADH ⟶ NAD

xx Beier UH, Angelin A, Akimova T, Wang L, Liu Y, Xiao H, Koike MA, Hancock SA, Bhatti TR, Han R, Jiao J, Veasey SC, Sims CA, Baur JA, Wallace DC, Hancock WW (2015) Essential role of mitochondrial energy metabolism in Foxp3⁺ T-regulatory cell function and allograft survival. FASEB J 29:2315-26. - »Bioblast link«

S7.4

xx Howie D, Waldmann H, Cobbold S (2014) Nutrient sensing via mTOR in T cells maintains a tolerogenic microenvironment. Front Immunol 5:409. - »Bioblast link«

S7.4

xx Murray AJ (2009) Metabolic adaptation of skeletal muscle to high altitude hypoxia: how new technologies could resolve the controversies. Genome Med 1:117. - »Bioblast link«

S7.4

xx Prasun P (2020) Role of mitochondria in pathogenesis of type 2 diabetes mellitus. J Diabetes Metab Disord 19:2017-22. - »Bioblast link«

S7.4

xx Steiner JL, Lang CH (2017) Etiology of alcoholic cardiomyopathy: Mitochondria, oxidative stress and apoptosis. Int J Biochem Cell Biol 89:125-35. - »Bioblast link«

S7.4

xx Tirichen H, Yaigoub H, Xu W, Wu C, Li R, Li Y (2021) Mitochondrial reactive oxygen species and their contribution in chronic kidney disease progression through oxidative stress. Front Physiol 12:627837. - »Bioblast link«

S7.4

Supplement 8. NADH + H ⟶ NAD⁺ + 2e^-

xx Vartak R, Porras CA, Bai Y (2013) Respiratory supercomplexes: structure, function and assembly. Protein Cell 4:582-90. - »Bioblast link«

Fig. 1 of Vartak et al (2013): The misconstrued charge on FAD in FADH₂ → FAD⁺ may explain the explicit one-electron (1e) transfer shown for the 2-electron transfer from FADH₂.

S8

Supplement 9. NADH + H⁺ ⟶ NADH

xx Cadonic C, Sabbir MG, Albensi BC (2016) Mechanisms of mitochondrial dysfunction in Alzheimer's disease. Mol Neurobiol 53:6078-90. - »Bioblast link«

S9

Supplement 10. NADH₂ ⟶ NAD⁺

xx Papa S, Petruzzella V, Scacco S (2007) Electron transport. Structure, redox-coupled protonmotive activity, and pathological disorders of respiratory chain Complexes. Springer, Boston, MA. In: Lajtha A, Gibson GE, Dienel GA (eds) Handbook of neurochemistry and molecular neurobiology:93–118. - »Bioblast link«

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Supplement 11. NADH ⟶ for CI, but NAD⁺ ⟶ NADH + H⁺ for TCA cycle DH

xx DiMauro S, Rustin P (2009) A critical approach to the therapy of mitochondrial respiratory chain and oxidative phosphorylation diseases. Biochim Biophys Acta 1792:1159-67. - »Bioblast link«

S11

Supplement 12. Reverse

xx Grandoch M, Flögel U, Virtue S, Maier JK, Jelenik T, Kohlmorgen C, Feldmann K, Ostendorf Y, Castañeda TR, Zhou Z, Yamaguchi Y, Nascimento EBM, Sunkari VG, Goy C, Kinzig M, Sörgel F, Bollyky PL, Schrauwen P, Al-Hasani H, Roden M, Keipert S, Vidal-Puig A, Jastroch M5, Haendeler J, Fischer JW (2019) 4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue. Nat Metab 1:546-59. - »Bioblast link«

NADH is shown as the product of the reaction catalyzed by CI in respiration. This error is rare in the literature, but comparable to the error frequenty encountered when FADH₂ is shown as the substrate of CII.

S12

xx Hunt KA, Jennings RM, Inskeep WP, Carlson RP (2018) Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community. PLoS Comput Biol 14:e1006431. Nat Metab 1:546-59. - »Bioblast link«

NADH is shown as the product of the reaction catalyzed by CI in respiration.

S12

xx Zhao H, Li Y (2021) Cancer metabolism and intervention therapy. Mol Biomed 2:5. - »Bioblast link«

S12

Supplement 13. Cytochrome b₆f Complex: NADP⁺ + H⁺ ⟶ NADPH

xx Baniulis D, Yamashita E, Zhang H, Hasan SS, Cramer WA (2008) Structure-function of the cytochrome b6f complex. Photochem Photobiol 84:1349-58. - »Bioblast link«

S13

xx Hasan SS, Cramer WA (2012) On rate limitations of electron transfer in the photosynthetic cytochrome b6f complex. Phys Chem Chem Phys 14:13853-60. - »Bioblast link«

S13

xx Liguori N, Croce R, Marrink SJ, Thallmair S (2020) Molecular dynamics simulations in photosynthesis. Photosynth Res 144:273-95. - »Bioblast link«

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Supplement 14. Other

xx Michelet L, Zaffagnini M, Morisse S, Sparla F, Pérez-Pérez ME, Francia F, Danon A, Marchand CH, Fermani S, Trost P, Lemaire SD (2013) Redox regulation of the Calvin-Benson cycle: something old, something new. Front Plant Sci 4:470. - »Bioblast link«

xx Lauterbach L, Lenz O, Vincent KA (2013) H₂-driven cofactor regeneration with NAD(P)⁺-reducing hydrogenases. FEBS J 280:3058-68. - »Bioblast link«

## Li F, Hinderberger J, Seedorf H, Zhang J, Buckel W, Thauer RK (2008) Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri. J Bacteriol 190:843-50. - »Bioblast link«

## Reeve HA, Lauterbach L, Lenz O, Vincent KA (2015) Enzyme-modified particles for selective biocatalytic hydrogenation by hydrogen-driven NADH recycling. ChemCatChem 7:3480-7. - »Bioblast link«

## Liang J, Huang H, Wang S (2019) Distribution, evolution, catalytic mechanism, and physiological functions of the flavin-based electron-bifurcating NADH-dependent reduced ferredoxin: NADP+ oxidoreductase. Front Microbiol 10:373. - »Bioblast link«