Description

Pathway control states (synonymous with ETS substrate control states) are obtained in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, tissue homogenate) by depletion of endogenous substrates and addition to the mitochondrial respiration medium of fuel substrates (CHNO) activating specific mitochondrial pathways. Mitochondrial pathway control states, mtPCS, have to be defined complementary to mitochondrial coupling control states. Coupling states (LEAK, OXPHOS, ETS) require electron transfer system competent substrate control states, including oxygen supply. Categories of SUIT protocols are defined according to mitochondrial pathway control states. » MiPNet article

Abbreviation: PCS, mtPCS

Reference: Gnaiger 2009 Int J Biochem Cell Biol, Gnaiger 2014 MitoPathways, Categories of SUIT protocols

MitoPedia concepts: MiP concept, Respiratory state, SUIT concept, SUIT state

MitoPedia methods: Respirometry

MitoPedia topics: Substrate and metabolite

Pathway control states

Gnaiger E (2016) Pathway control states. Mitochondr Physiol Network 2016-08-12, edited 2016-11-08.

OROBOROS (2016) MiPNet

Abstract: Pathway control states are defined in mitochondrial preparations complementary to coupling control states in mitochondrial physiology.

• O2k-Network Lab: AT Innsbruck Gnaiger E

Labels:

Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP

Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. Pathway: F, N, S, Gp, CIV, NS, Other combinations HRR: Theory

ETS pathway types

ETS pathway types are linked to ETS substrate types in mitochondrial SUIT protocols. Mitochondrial pathways are stimulated by fuel substrates (CHNO) feeding electrons into the electron transfer system (ETS) at different levels of integration and in the presence or absence of inhibitors acting on specific enzymes in these pathways. Distinction of five ETS pathway types provides the rationale for defining categories of SUIT protocols.

ETS pathway type 1 is stimulated by artificial electron donors (e.g. TMPD, Tm) essentially bypassing the ETS, reducing cytochrome c and feeding electrons to cytochrome c oxidase (CIV) or alternative oxidases (single enzymatic step), with oxygen as the terminal electron acceptor.
ETS pathway type 2 is stimulated by duroquinol (DQ) feeding electrons into Complex III (CIII) with further electron transfer to CIV and oxygen.
ETS pathways type 3 feed electrons from NADH, FADH₂, succinate, glycerophosphate into respiratory complexes directly upstream of the Q-junction. NADH is the substrate of Complex I (CI). FADH₂ is the substrate of electron transferring flavoprotein (CETF) localized on the inner side of the inner mt-membrane. Succinate is the substrate of succinate dehydrogenase (SDH, CII) localized on the inner side of the inner mt-membrane. Glycerophosphate is the substrate of glycerophosphate dehydrogenase complex (CGpDH) localized on the outer face of the inner mt-membrane. Choline is the type 3 substrate of choline dehydrogenase.
ETS pathway type 4 feeds electrons into dehydrogenases and enzyme systems upstream of the type 3 pathway level. Electron transfer from type 4 substrates (N) converges at the N-junction. Representative N-junction substrates are pyruvate, glutamate and malate, and also citrate, oxoglutarate and others. The corresponding dehydrogenases (PDH, GDH, MDH and mtME; IDH, OgDH) generate NADH, the substrate of Complex I (CI). Fatty acids supporting converging electron transfer to the F-junction might also considered as type 4 substrates. However, the requirement of a combined operation of the F-junction and N-junction puts type F substrates to a higher level of pathway integration.
ETS pathway type 5 feeds electrons into dehydrogenases and enzyme systems upstream of the type 3 pathway level with an obligatory combination of the F-junction and N-junction. F-junction substrates are fatty acids involved in β-oxidation, generating (enzyme-bound) FADH₂, the substrate of electron transferring flavoprotein (CETF). Succinate does not belong to the type 4 substrates, since FADH₂ is the product of CII, whereas FADH₂ is the substrate of CETF. Fatty acid oxidation (FAO) not only depends on electron transfer through the F-junction (which is typically rate-limiting) but simultaneously generates NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). In addition and independent of this source of NADH, the N-junction substrate malate is required as a co-substrate for FAO in mt-preparations, since accumulation of AcetylCoA inhibits FAO in the absence of malate. Malate is oxidized in a reaction catalyzed by malate dehydrogenase to oxaloacetate (yielding NADH), which then stimulates the entry of AcetylCo into the TCA cycle catalyzed by citrate synthase.

ETS substrate types on different pathway levels

ETS substrates type 5 on the pathway level of converging FADH₂ and NADH-linked dehydrogenases, including beta-oxidation and segments of the TCA cycle:

F: F-junction substrates, FADH₂-linked, fatty acids (FAO)

ETS substrates type 4 on the pathway level of converging NADH-linked dehydrogenases, including the TCA cycle:

N: N-junction substrates, NADH-linked (and hence downstream 'CI-linked')

ETS substrates type 3 on the pathway level of electron transfer complexes converging at the Q-junction:

Q: Q-junction substrates

NADH, substrate of CI
FADH₂, substrate of CETF
S: Succinate, substrate of CII
Gp: Glycerophosphate, substrate of CGpDH
Choline: substrate of choline dehydrogenase

ETS substrates type 1 on the single step level of cytochrome c oxidase (CIV), the terminal step in the aerobic electron transfer system:

Tm: Artificial electron transfer susbstrate TMPD (Tm) maintained in a reduced state by ascorbate (As) and reducing cytochrome c as the substrate of CIV.

Terminology: from CI- and CII-linked to N and S

CI and CII are abbreviations for enzymes, respiratory Complex I (CI) and Complex II (CII).
In Gnaiger 2009, the 2007-2014 editions of MitoPathways and many previous publications, CI-linked respiration has been used to indicate respiration supported by NADH-generating substrates, N (pyruvate, glutamate, malate, or other ETS-competent N-type substrate combinations). In this N-type pathway, electron transfer converges from dehydrogenases to the NADH-junction, and from NADH through CI to the Q-junction, with further electron transfer through CIII and CIV to oxygen. Similarly, CII-linked respiration indicates respiration supported by succinate, S, and electron transfer through CII to the Q-junction, with further electron transfer through CIII and CIV to oxygen.
Convergent electron flow from a combination of NADH-generating substrates (N) and succinate (S) has been indicated as CI&II-linked respiration (Gnaiger 2014 MitoPathways), synonymous with NS. The symbol '&' in CI&II helps to distinguish CI&II as the measured flux in the presence of both NADH-linked substrates and succinate, in contrast to CI+CII (or N+S) as the algebraic sum of fluxes measured separately in the N- versus S-patwhay control states.
Simplification: NS denotes flux under the simultaneous control of the NADH- and succinate-linked pathways (NS-substrate cocktail), in contrast to N+S as the algebraic sum of the fluxes controlled by the two pathways separately.

ETS competent pathway control states

Coupling states (LEAK, OXPHOS, ETS) require electron transfer system (ETS) competent pathway states based on external substrate supply, including sufficient oxygen supply. ETS competence of external substrates requires (i) transport of substrates across the inner mt-membrane or oxidation by dehydrogenases located on the outer face of the inner mt-membrane (e.g. glycerophosphate dehydrogenase complex, CGpDH), (ii) oxidation in the mt-matrix (TCA cycle dehydrogenases and other matrix dehydrogenases, e.g. mtGDH) or on the inner face of the inner mt-membrane (succinate dehydrogenase), (iii) oxidation of substrates without accumulation of inhibitory endproducts (e.g. oxaloacetate inhibiting succinate dehydrogenase; NADH and oxaloacetate inhibiting malate dehydrogenase), and (iv) electron transfer through the membrane-bound ETS (mETS). Endproducts must be either easily exported from the matrix across the inner mt-membrane (e.g. malate formed from succinate via fumarate), or metabolized in the TCA cycle (e.g. malate-derived oxaloacetate forming citrate in the presence of external pyruvate&malate).

Single pathway control states

Single pathway control states are pathway control states for selective entry of electron transfer into the Q-junction through one particular respiratory complex; for instance N-respiration through CI (PM; GM; PGM with or without malonic acid: Gnaiger 2014 MitoPathways Chapter 3), S-pathway control state with e-input into Q through CII, CIV (Tm: MiPNet06.06_Chemical O2 background).

Further details, see Categories of SUIT protocols.

Multiple pathway control states

Multiple pathway control states are pathway control states obtained in intact cells respiring on endogenous substrates or in media with physiological exogenous substrates, or designed for reconstitution of TCA cycle function in isolated mitochondria, permeabilized cells or permeabilized tissues. In all cases, electron flow converges at the Q-junction with multiple entry sites of NS-electron transfer through CI&II, FNS through FAO&CI&II, NSGp through CI&II&GpDH.

Further details » Categories of SUIT protocols

Pathway versus kinetic substrate control

Control by substrate type: pathway control states

A: Intact cells

Endogenous pathway control: In intact cells, ce, endogenous organic carbon substrates are mobilized in the cytosol as intermediary metabolites transported across the inner mitochondrial membrane and thus exerting control over mitochondrial respiration. If no organic carbon substrates are supplied in the incubation medium, then substrate control is entirely endogenous. Long-term incubation under such conditions leads to progressive depletion of endogenous substrates.
Exogenous pathway control: Cells are grown in complex culture media with a variety of organic carbon substrates, and different exogenous pathway control states are achieved by variation of these substrates. Long-term incubation in closed systems without exchange of culture medium leads to progressive depletion of exogenous substrates. Incubation of cells in simple media allows for sequential titration of specific carbon substrates (e.g. glucose or fructose; lactate or glutamate; fatty acids) for the study of exogenous pathway control of respiration.

B: Mitochondrial preparations

Specific substrate-inhibitor combinations are selected to establish pathway states for (i) stimulating defined segments of the electron transfer system, or (ii) reconstitution of TCA cycle function.

Pathway control states with electron gating: Specific substrate-inhibitor combinations are applied for selectively stimulating electron entry from N-junction substrates through CI, S-pathway control state through CII, or other substrates feeding additional branches converging at the Q-junction, particularly F-type (fatty acid oxidation and Gp (glycerophosphate). The most commonly applied pathway states select for N-electron input through Complex I (pyruvate&malate, PM; glutamate&malate, GM), S-pathway control state ([[Complex II]-pathway to Q]: succinate and rotenone, SRot), or Complex IV electron input (CIV: ascorbate&TMPD(Ama)).
Physiological pathway control states: Reconstitution of TCA cycle function requires NS-substrate combinations, such as PMS, GMS, GS, PGMS, or PGS, applied simultaneously without inhibitor of any respiratory complexes.

Control by substrate concentration: kinetic control states

Kinetic substrate or adenylate control: Kinetic studies with variation of a specific substrate (reduced substrate supplying electrons to the ETS; ADP, Pi; O₂; cytochrome c) are analyzed by kinetic functions (e.g. hyperbolic), yielding kinetic parameters, such as J_max, K_m', c₅₀ [µM], or p₅₀ [kPa].
Kinetic inhibitor control: Kinetic studies with variation of a specific inhibitor yield apparent kinetic constants, such as the K_I'.