An overview of NADH, FADH₂, NADPH, Acyl-CoA & Acetyl-CoA used in Metabolism
By Curtis Bustos

To begin, what are coenzymes? Coenzymes are a type of cofactor. What are cofactors? Cofactors help enzymes perform their functions. Some enzymes will not work without being associated with a specific cofactor. Cofactors are sometimes classified as an "enzyme's teeth" because it helps grab and hold on to the substrate. By this, cofactors are not considered as substrates.

Key Terms of redox-coenzymes:

NAD⁺ = Nicotinamide Adenine Dinucleotide

FAD⁺ = Flavin Adenine Dinucleotide

NADP⁺ = Nicotinamide Adenine Dinucleotide Phosphate

CoA = Coenzyme Adenine

Acyl-CoA = Acyl-Coenzyme Adenine

Acetyl-CoA = Acetyl-Coenzyme Adenine

It seems there are many important molecules with adenine as part of their structure such as ATP. Why adenine and not some other high-energy producing molecule? The answer may be due to evolutionary economy or an evolutionary advantage it has from its ability to perform multiple roles. 

What is the difference between NADH & NADPH?

The redox chemistry doesn’t really change with NADH & NADPH. However, the phosphate group attached to NADPH allows for enzymes to differentiate between the two coenzymes. This allows different pathways for anabolic and catabolic metabolism. For example, NADP⁺ is a coenzyme in the pentose-phosphate-pathway and is used during anabolic metabolism whereas NAD⁺ acts as an electron acceptor and is used in catabolic pathways such as glycolysis and the citric acid cycle.

What is the difference between acyl-CoA and acetyl-CoA? It simply depends on what CoA attaches itself to. If CoA attaches itself to an acyl-group (fatty acid), it will be called acyl-CoA and can be used during β oxidation. However, if CoA attaches itself to an acetyl group (COCH₃), it will be called acetyl-CoA.

Where does FADH₂ make an appearance?

FADH₂ is found in the citric acid cycle, the mitochondria during β oxidation, and in the electron transport chain (ETC) during oxidative phosphorylation.

Below is a list of co-enzymes and their half-reactions where E’^o represents a REDUCTIVE POTENTIAL (measured in volts) that simply gauges a molecule’s tendency to be reduced or oxidized.

NAD⁺ (oxidized)  à NADH (reduced)

Half Reaction = NAD⁺ + H⁺ + 2e^- à lactate^-    (E’^o = -0.320)

FAD⁺ (oxidized) à  FADH à   FADH₂ (reduced)

Half Reaction = FAD + 2H⁺ + 2e^- à FADH₂    (E’^o = -0.219)

* This is the value for free FAD; FAD bound to a specific flavoprotein has a different E’^o that depends on its protein environment.

NADP⁺ (oxidized)  à  NADPH (reduced)

Half Reaction = NADP⁺ + H⁺ + 2e^- à NADPH   (E’^o = -0.324)

NADH, NADPH, FADH₂, CoA, Acetyl-CoA & Acyl-CoA in their molecular forms:

A closer view of NADH & NADPH in its oxidized and reduced forms:

Since FAD⁺ is able to carry two H-atoms, the reaction pathway will consist of two steps:

CoA (HS-CoA) vs. Acetyl-CoA (CH₃-CO-O-S-CoA) vs. Acyl-CoA (CH₃-(CH₂)_n-CO-SCoA):

Next, we are going to take an overview-approach to see how these coenzymes apply to metabolism. Before we begin, you should embed this flow-chart into your memory and refer to it whenever you get stuck:

Be able to apply coenzymes to other half-reactions for specific functions during metabolism. NOTE (x2) will reflect that there are two GAP or pyruvate molecules contributing to the metabolic pathways listed below. 

Glycolysis: NET = NADH (x2)

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NAD+	GAP à 1,3-BPG NAD+ à NADH (x2)	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Step 6 during glycolysis.	NAD+ is being reduced during this process.

Gluconeogenesis: NET = NAD⁺ (x2)

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NADH	1,3-biphoshphoglycerate       à glyceraldehyde-3-phosphate (GAP) NADH (x2) à NAD+ (x2)	Occurs during gluconeogenesis.	NADH is being oxidized during this process.

Pyruvate à Lactate: NET = NAD⁺ (x2)

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NADH	Pyruvate à Lactate NADH à NAD+ (x2)	Under anaerobic conditions.	NAD+ is being oxidized during this process.

Pyruvate à Acetyl CoA: NET = NADH (x2)

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NAD+	Pyruvate à Acetyl CoA NAD+ à NADH (x2)	Pyruvate Dehydrogenase step.	NAD+ is being reduced during this process.

β Oxidation:

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NAD+	NAD+ à NADH	β oxidation in mitochondria	NAD+ is being reduced during this process.
FAD+	FAD+ à FADH2	β oxidation in mitochondria	FAD+ is being reduced during this process.
CoA	Triacylglycerol à acetyl-chain CoA àAcetyl-CoA	β oxidation in mitochondria	NA
CoA	Triacylglycerol à acyl-chain CoA àAcyl-CoA	β oxidation in mitochondria	NA

Citric Acid Cycle: NET = NADH (3x2 = 6) & FADH₂ (x2)

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NAD+	D-Isocitrate à a-ketogluterate NAD+ à NADH (x2)	Tricarboxylic Acid Cycle (TCA) Step 3	NAD+ is being reduced during this process.
NAD+	a-ketogluterate à Succinyl-CoA NAD+ à NADH (x2)	Tricarboxylic Acid Cycle (TCA) Step 4	NAD+ is being reduced during this process.
NAD+	Malate àOxaloacetate NAD+ à NADH (x2)	Tricarboxylic Acid Cycle (TCA) Step 8	NAD+ is being reduced during this process.
FAD+	Substrate-level phosphorylation à Dehydrogenation FAD+ à FADH2 (x2)	Cycle (TCA) Step 6	FAD+ is being reduced during this process.

For fun, fill in the gaps on the citric acid cycle below:

  

Oxidative Phosphorylation: NET 1NADH = 2.5 ATP, 1FADH₂ = 1.5 ATP

Coenzyme	Half-reactions (redox reactions are half oxidized and half reduced)	Chemical process or step being applied.	Is the coenzyme being oxidized (loss of electrons) or reduced (gain of electrons)?
NADH	NADH dehydrogenase NADH à NAD+ (contributes to 10H+ ions in the intermembrane space)	Complex I in the ETC.	NADH is being oxidized during this process.
FADH2	Succinate dehydrogenase FADH2 à FAD+ (contributes to 6H+ ions in the intermembrane space)	Complex II in the ETC.	FADH2  is being oxidized during this process.