Complex II ambiguities-FADH2 in the electron transfer system

Abstract

The prevailing notion that reduced cofactors NADH and FADH₂ transfer electrons from the tricarboxylic acid cycle to the mitochondrial electron transfer system creates ambiguities regarding respiratory Complex II (CII). CII is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the electron transfer system of the mitochondrial inner membrane feeding electrons into the coenzyme Q-junction. The succinate dehydrogenase subunit SDHA of CII oxidizes succinate and reduces the covalently bound prosthetic group FAD to FADH₂ in the canonical forward tricarboxylic acid cycle. However, several graphical representations of the electron transfer system depict FADH₂ in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH₂ from the β-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature and educational resources call for quality control, to secure scientific standards in current communications of bioenergetics, and ultimately support adequate clinical applications. This review aims to raise awareness of the inherent ambiguity crisis, complementing efforts to address the well-acknowledged issues of credibility and reproducibility.

Keywords: Complex II; coenzyme Q; electron transfer system; fatty acid oxidation; flavin adenine dinucleotide; succinate dehydrogenase; tricarboxylic acid cycle.

Figure 1

Complex II (SDH) integrates H⁺-linked electron transfer in the TCA cycle (matrix-ETS) and the electron transfer system (membrane-ETS) of the mt-inner membrane (mtIM). A, NADH+H⁺ and (B) Succinate are substrates of 2{H⁺ + e^-} transfer to the prosthetic groups FMN and FAD as the corresponding electron acceptors in CI and CII, respectively. C, symbolic representation of ETS pathway architecture. Electron flow converges at the N-junction, NAD⁺+2{H⁺ + e^-} → NADH+H⁺, and from NADH+H⁺ and succinate S at the Q-junction, UQ+2{H⁺ + e^-} → UQH₂. CIII passes electrons to cytochrome c and in CIV to molecular O₂, 2{H⁺ + e^-}+0.5 O₂ ⇢ H₂O. D, NADH and NAD⁺ cycle between different matrix-dehydrogenases and CI, whereas FAD and FADH₂ remain permanently bound within the same CII-enzyme molecule during the catalytic cycle. Succinate and fumarate indicate the chemical entities irrespective of ionization, whereas charges are shown in NADH (uncharged), NAD⁺, and H⁺. Joint pairs of half-circular arrows distinguish the chemical reaction of electron transfer 2{H⁺ + e^-} to CI and CII from vectorial H⁺ translocation across the mtIM (H⁺_neg → H⁺_pos). CI, CIII, and CIV pump hydrogen ions from the negatively (neg; yellow, mt-matrix) to the positively charged compartment (pos; grey, intermembrane space). E, Iconic representation of SDH subunits. SDHA catalyzes the oxidation succinate → fumarate + 2{H⁺ + e^-} and reduction FAD+2{H⁺ + e^-} → FADH₂ in the soluble domain of CII. The iron–sulfur protein SDHB transfers electrons through Fe-S clusters to the mtIM domain where ubiquinone UQ is reduced to ubiquinol UQH₂ in SDHC and SDHD.

Figure 2

Electron transfer to CI and CII. Zoom into figures of ref. (45). A, H⁺ (marked) is shown to be produced in H⁺-linked electron transfer instead of being consumed (cf. Fig. 1A). B, Marked quote inserted from the legend to Fig. 10.9 of ref. (45).

Figure 3

ComplexII ambiguities. FADH₂ is depicted as (1) product and (2) substrate of Complex II by (A) (4), (B) (80), as in ref. (48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 104, 192, 193, 194, 199, 201, 202, 203, 204, 205, 206, 209, 210, 211, 213, 215, 216, 217, 218, 219, 220, 222, 225, 226, 227, 231, 233, 236, 239, 240, 241, 245, 271-280, 285, 291, 292, 293, 294, 295, 304, 305, 306, 308, 311, 312, 314, 320, 330). NADH and NAD⁺ cycle between different types of enzymes (yellow circle), in contrast to the FAD/FADH₂ cycle located within the same enzyme molecule (SDH and CII are synonyms). C, from ambiguous NADH-FADH₂ analogy (73) to (D) graphical misconception (58), as in ref. (100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240). NADH and FADH₂ at the doors of CI and CII, respectively, shown by (E) an international team (169) and (F) an international consortium suggesting guidelines (198); FADH₂ cannot enter – it functions always inside CII like FAD which receives electrons from the substrate succinate. G, the redox reaction of the flavin adenine dinucleotide is copied from the nicotinamide adenine dinucleotide with an unjustified indication of 2H⁺ formation in the mt-matrix, confusing in the context of the protonmotive force. The figure from ref. (264) is similar or identical to zooms into 33 figures from ref. (49, 57, 70, 72, 133, 154, 186, 244, 246, 249, 251, 252, 253, 254, 255, 257, 262, 263, 264, 267, 269, 270, 272, 273, 275, 276, 277, 278, 279, 280, 317, 319). H, the CII ambiguity in FADH₂→FAD+2H⁺ (242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280) fires back at the CI-catalyzed reaction when NAD⁺ is shown like FAD as NAD without charge (245, 271). I, the NADH→NAD⁺ analogy is taken to the level of copying a charge to FAD⁺ ((288), as in (281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319)) or (J) FADH⁺ ((331), as in (332)). K, exponential increase of publications with graphical Complex II ambiguities, 2001 to October 2023. Open symbol: the count of 46 publications in 2023 was adjusted for the full year by a multiplication factor of 12/10. Asterisc: zero count in 2004 set at 0.1 for exponential fit. N = 312 is the number of publications found with graphical CII ambiguities (Table 2).

Figure 4

Convergent electron transfer into the NAD junction, ETF junction, and Q-junction indicated by convergent arrows, without showing the alignment of supercomplexes. Inter-membrane space (interM) indicated in grey and mt-matrix in yellow-orange. A, convergent FAD-linked electron transfer into the ETF junction as the first step in β-oxidation from very long- and long-chain acyl-CoA dehydrogenases (ACADS, membrane-bound), medium-, and short-chain ACADs including short/branched-ACAD (SBCAD) and Complex I assembly factor ACAD9; in branched-chain amino acid oxidation from glutaryl-CoA DH (GCDH), SBCAD, isobutyryl-CoA DH (IBDH) and isovaleryl-CoA DH (IVDH); in choline metabolism from dimethylglycine DH (DMGDH) and sarcosine DH (SARDH); and from acyl-CoA DH family members 10 and 11 (ACAD10, ACAD11) and D-2-hydroxyglutarate DH (D2HGDH). References (357, 360, 361). ETF is the redox shuttle feeding electrons into the membrane-bound electron transferring flavoprotein Complex (CETFDH on the matrix side of the mtIM) and further into Q. Steps two to four in β-oxidation of long- and medium-chain fatty acids are catalyzed by trifunctional protein (TFP, membrane-bound). Step three reduces NAD⁺ to NADH + H⁺, feeding electrons into the NAD-cycle, catalyzed by TFP and short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). B, N: N-pathway through Complex I (CI; see Fig. 1). F: F-pathway of fatty acid oxidation through the β-oxidation cycle (β-ox) with ACADs binding noncovalently FAD; converging electron transfer through ETF to CETFDH, and dependence on the N-pathway. S: S-pathway through CII. Gp: Gp-pathway through mt-glycerophosphate DH Complex (CGpDH on the inter-membrane side of the mtIM) oxidizing glycerophosphate to dihydroxyacetone phosphate (DHAP) in the inter-membrane space.

Figure 5

WhenFADH₂is erroneously shown[1]as a substrate of CII, then[2]a dubious role of CII in oxidation of FADH₂from β-oxidation is suggested as a consequence. Zoom into figures (A) (52); (B) (71); (C) (225); (D) (97); (E) (220); (F) (227); (G) (283, 299); (H) (293); (I) (218); (J) paradoxical oxidation of FADH₂ and NADH in β-oxidation and reduction by CI of NAD (NAD⁺) from β-oxidation (56); (K) (295); (L) (62); (M) (291); (N) (83); (O) (349, 352, 353, 354, 355); (P) (204); (Q) (192); (R) (51) as in (66); (S) (348); (T) (350); (U) (351).

Figure 6

Ambiguousconclusions on a direct role of CII in the oxidation of glycerophosphate and other “FADH₂-linked” pathways, analogous to false representations of CII involved in fatty acid oxidation (Fig. 5). FADH₂[1] formed in the mt-matrix as a product of the TCA cycle and [2] feeding into CII: (A) (75); among 312 examples of CII-ambiguities; and FADH₂[1] formed in the mt-matrix from (B) GPO1 (90), (C) GPD2 (370), or (D) GPDH (365), and [2] feeding into the ETS. GPO1, GPD2, and GPDH indicate the respiratory Complex CGpDH on the inter-membrane side of the mtIM (Fig. 4B). E–G, the glyceraldehyde-3-phosphate shuttle is erroneously shown to transfer the redox pair FAD/FADH₂ (not FADH/FADH₂) into the mt-matrix (358, 366, 370). H, the figure (345) suggests that electron transfer into the Q-junction occurs from a common FADH₂ pool generated by CII and CGpDH (GPD2). I and J, The FAD/FADH₂ redox system is implicated in various electron transfer pathways independent of CII, but the CII ambiguity does not make this sufficiently clear (59, 84). FADH₂ must be distinguished as a covalently bound prosthetic group of CII from a coenzyme that is attached loosely and transiently to an enzyme (385).