12.4: Biological Oxidation-Reduction Reactions

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

NAD⁺ and FAD as Biological Oxidizing Agents

• Compare NAD⁺ and FAD as biological oxidizing agents in terms of electron transfer mechanism (hydride vs. single-electron transfer), binding affinity to enzymes, and the types of oxidation reactions each is suited to catalyze, using standard reduction potentials to justify the distinction.
• Explain how the microenvironment of a tightly bound FAD cofactor can shift its standard reduction potential over a wide range, drawing an analogy to how protein environment affects amino acid pKa values.

Oxygenases: Incorporating Oxygen into Substrates

• Distinguish monooxygenases from dioxygenases in terms of the fate of both oxygen atoms from O₂, and provide a biological example of each, explaining the physiological significance of the hydroxylation reaction.
• Describe the catalytic cycle of cytochrome P450, including the roles of the heme iron, NADPH-cytochrome P450 reductase, and the reactive Fe⁴⁺-oxo intermediate, and explain how this enzyme both detoxifies nonpolar substrates and activates procarcinogens.
• Explain how cytochrome P450 gene expression is induced by nonpolar aromatic ligands such as dioxin through the AhR–Arnt transcription factor pathway, and describe the consequences of this induction for estrogen signaling and cancer risk.
• Describe the cyclooxygenase (COX) reaction, including how two molecules of dioxygen are incorporated into arachidonic acid to form PGG2 and the role of a tyrosine free radical in initiating the reaction.

Oxidases and the Broader Framework of Biological Oxidation

• Distinguish oxidases from oxygenases by explaining that oxidases transfer electrons from organic substrates to dioxygen via intermediate carriers — producing superoxide, hydrogen peroxide, or water — without incorporating oxygen into the organic substrate.
• Compare the functional roles of heme in hemoglobin/myoglobin, cytochrome C, and cytochrome P450, explaining how differences in axial ligands and protein microenvironment account for their distinct activities (O₂ transport, electron shuttling, and substrate hydroxylation, respectively).

General Oxidizing Agents

Before considering common biological oxidizing agents, look back at the ones you saw in other chemistry classes. Oxidizing agents are required to oxidize organic molecules. In chemistry labs, you likely never used dioxygen as an oxidizing agent. It is difficult to limit oxidation with dioxygen. In addition, side reactions are likely given the nature of the reactive oxygen reduction products. The mechanisms of combustion reactions of organic molecules with dioxygen to produce carbon dioxide and water are complicated. Table \(\PageIndex{1}\) shows some key steps in the combustion of methane.

Initiation

\[\ce{CH4 → CH3^{.} + H^{.}} \nonumber\]

\[\ce{O2 → 2 O^{.}} \nonumber\]

Propagation

\[\ce{CH4 + H^{.} → CH3^{.} + H2} \nonumber\]

\[\ce{CH4 + HO^{.} → CH3^{.} + H2O} \nonumber\]

\[\ce{CH3^{.} + O^{.} → CH2O + H^{.}} \nonumber\]

\[\ce{CH2O + HO^{.} → CHO^{.} + H2O} \nonumber\]

\[\ce{CH2O + H^{.} → CHO^{.} + H2} \nonumber\]

\[\ce{CHO^{.} → CO + H^{.}} \nonumber\]

\[\ce{CO + HO^{.} → CO2 + H^{.}} \nonumber\]

Branching

\[\ce{H^{.} + O2 → HO^{.} + O^{.}} \nonumber\]

Termination

\[\ce{H. + R^{.} + M → RH + M^{*}} \nonumber\]

In chemistry, other oxidizing agents are often used, including permanganate and chromate. Their mechanism of action is illustrated in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Mechanism of oxidation of organic molecules by permanganate and chromate

Oxygen can often be inserted into a molecule in a nonoxidative process by the hydration of an alkene to an alcohol (a readily reversible reaction), which can then be oxidized to either an aldehyde/ketone or a carboxylic acid using an appropriate oxidizing agent.

Most biological oxidation reactions (such as those found in glycolysis, the Krebs cycle, and fatty acid oxidation) do not use dioxygen as the immediate oxidizing agent. Instead, they use nicotinamide adenine dinucleotide (NAD⁺) or flavin adenine dinucleotide (FAD) as oxidizing agents, which are reduced. Enzymes that use these oxidizing agents are usually called dehydrogenases. Dioxygen can also introduce oxygen atoms into biological molecules in oxidative reactions. Enzymes that introduce one oxygen atom of dioxygen into a molecule (and the other oxygen into water) are called monooxygenases. (Note: some monooxygenases that hydroxylate biomolecules are called hydroxylases.) Those that introduce both atoms of dioxygen into a substrate are called dioxygenases. These oxygenases are not usually used to oxidize organic molecules for energy production. Instead, they introduce O atoms for other reasons, including increasing the solubility of nonpolar aromatics to facilitate secretion and producing new molecular species with different biological activities. Finally, dioxygen can oxidize biological molecules without adding oxygen atoms to the substrate. Instead, electrons lost from the oxidized substrate are passed via intermediate electron carriers to dioxygen, which gets reduced to superoxide (if one electron is added), hydrogen peroxide (if two electrons are added), or water (if 4 electrons are added). These enzymes are called oxidases. (Note: The letters oxi- or oxygen- are used in all the enzymes that use dioxygen as the oxidizing agent.)

In this chapter section, we will discuss biological oxidation reactions. Most introductory biochemistry texts don't treat oxidation reactions in a single cohesive chapter. Probably because of that, when I was learning biochemistry, I found the presentation of these different enzymes involved in redox reactions very confusing. Hopefully, this section will alleviate that problem. First, the chemistry of NAD⁺ and FAD will be discussed. Then, enzymes that use dioxygen in oxidative reactions (monooxygenases, dioxygenases, and oxidases) will be explored.

The Chemistry of NAD⁺ and FAD

NAD⁺ is a nicotinic acid or nicotinamide derivative, as illustrated in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Structure of niacin derivatives

It and its reduction product, NADH, exist in the cells as interconvertible members of a pool whose total concentration does not vary significantly with time. Hence, if NAD⁺ is oxidizing carbohydrates and lipids to produce energy in the form of ATP, levels of NAD⁺ would begin to fall as NADH rises. A mechanism must be present to regenerate NAD⁺ from NADH if oxidation continues. As we will see later, this happens in the muscle under anaerobic conditions (if dioxygen is lacking, as when you are running a 100 or 200 meter race, or if a saber-toothed tiger is chasing you) when pyruvate and NADH react to form lactate + NAD⁺. The reaction is shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Conversion of pyruvate to lactate

Under aerobic conditions (sufficient dioxygen available), NADH is reoxidized in the mitochondria via electron transport through various mobile electron carriers, which pass electrons to dioxygen (via the enzyme complex cytochrome c oxidase) to form water.

NAD⁺/NADH can undergo two-electron redox steps, in which a hydride is transferred from an organic molecule to the NAD⁺, with the electrons flowing to the positively charged nitrogen of NAD⁺, which serves as an electron sink. NADH does not react well with dioxygen, since single-electron transfers to/from NAD⁺/NADH produce free radical species that cannot be stabilized effectively. All NAD⁺/NADH reactions appear to involve 2-electron hydride transfers. Figure \(\PageIndex{5}\) shows both 1 and 2 electrons to NAD⁺.

Figure \(\PageIndex{4}\):

Figure \(\PageIndex{4}\): 1 and 2 electrons to NAD⁺

FAD (or flavin mononucleotide-FMN) and its reduction product, FADH₂, are derivatives of riboflavin, as shown in Figure \(\PageIndex{5}\).

 

Figure \(\PageIndex{5}\): Structures of riboflavin, FMN and FAD

FAD/FADH₂ differ from NAD⁺/NADH since they are bound tightly (K_D approx 10^-7 - 10^-11 M) to enzymes that use them. This is because FADH₂ is susceptible to reaction with dioxygen since FAD/FADH₂ can form stable free radicals via single-electron transfer. FAD/FADH₂ can undergo either one or two electron transfers. This is illustrated in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): 1 and 2 electrons reduction of FAD

FAD/FADH₂ are tightly bound to enzymes to control the nature of the oxidizing/reducing agents that interact with them. (i.e., so dioxygen in the cell won't react with them in the cytoplasm.) An enzyme with a tightly bound FAD that oxidizes a substrate would be inactive for further catalytic steps unless another oxidizing agent reoxidizes the bound FADH₂.

Dehydrogenases

These enzymes use NAD⁺/NADH or FAD/FADH₂ and are named for the oxidized substrate. For instance, in the reaction:

\[\ce{pyruvate + NADH <=> lactate + NAD^{+}} \nonumber\]

which is used to regenerate NAD⁺ under anaerobic conditions, the enzyme is named lactate dehydrogenase. As in acid/base reactions, when the preferred direction for the reaction (from a ΔG^o perspective) is from a stronger acid to a weaker (conjugate) acid, the preferred direction for a redox reaction is in the direction from a stronger to a weaker oxidizing/reducing agent. This can easily be determined from charts of standard reduction potentials and using the equation: ΔG^o = -nFE^o,

• where F is the Faraday constant (96,494 Coulombs/mol e^- = 96, 494 J/(V.mol) = 23.06 kcal/(V^.mol) or 96 kJ/(V^.mol). One Faraday is the charge per one mol of electrons).
• and E^o, the standard EMF or standard cell potential (total voltage at standard state conditions), which can be determined by adding the standard reduction potentials (E^o) for the two appropriate half-reactions after reversing the equation for the half-reaction that represents the oxidation. Hence E^o=E^o_reduction−E^o_oxidation.

When n=2 (number of electrons), which is common for oxidations of organic molecules,

ΔG^o (kcal/mol) = - 46.12E^o or approximately -50E⁰  (or -193E^o kJ/mol)

Notice that when E^o > 0, ΔG^o < 0, the reaction as written is favored under standard conditions. Note in the table below that many of the half-reactions involve protons. For biological reactions involving free protons, the standard-state concentration of protons is not 1 M, as for other solutes, but is defined as the hydronium ion concentration at pH 7.0. The E^o and ΔG^o values for the reactions involving hydrogen ions at a standard state of pH 7.0 are usually written as E^o' and ΔG^o'.

Table \(\PageIndex{2}\) below shows common standard reduction potentials.

Table \(\PageIndex{2}\): Standard reduction potentials for common biomolecules

oxidant	reductant	n (electrons)	E^o' (volts), 25^oC
Acetate + carbon dioxide	pyruvate	2	-0.70
succinate + CO2 + 2H^+	α−ketoglutarate + H2O	2	-0.67
acetate	acetaldehyde	2	-0.60
glycerate-3-P	glyceraldehyde-3-P + H2O	2	-0.55
O2	O2^-	1	-0.45
ferredoxin (ox)	ferredoxin (red)	1	-0.43
Carbon dioxide	formate	2	-0.42
2H^+	H2	2	-0.42
α-ketoglutarate + CO2 + 2H^+	isocitrate	2	-0.38
acetoacetate	β-hydroxybutyrate	2	-0.35
Cystine	cysteine	2	-0.34
Pyruvate + CO2	malate	2	-0.33
NAD^+ + 2H^+	NADH + H^+	2	-0.32
NADP^+ + 2H^+	NADPH + H^+	2	-0.32
FMN (enzyme bound)	FMNH2	2	-0.30
Lipoic acid, ox	Lipoic acid, red	2	-0.29
1,3 bisphosphoglycerate + 2H^+	glyceraldehyde-3-P + Pi	2	-0.29
Glutathione, ox	Glutathione, red	2	-0.23
FAD (free) + 2H^+	FADH2	2	-0.22
Acetaldehyde + 2H^+	ethanol	2	-0.20
Pyruvate + 2H^+	lactate	2	-0.19
Oxalacetate + 2H^+	malate	2	-0.17
α-ketoglutarate + NH4^+	glutamate	2	-0.14
FAD + 2H^+ (bound)	FADH2 (bound)	2	0.003-0.09
Methylene blue, ox	Methylene blue, red	2	0.01
Fumarate + 2H^+	succinate	2	0.03
CoQ (Ubiquinone - UQ) + H^+	UQH	1	0.031
UQ + 2H^+	UQH2	2	0.06
Dehydroascorbic acid	ascorbic acid	2	0.06
Ubiquinone; ox	red	2	0.10
Cytochrome b2; Fe^3^+	Cytochrome b2; Fe2+	1	0.12
Cytochrome c1; Fe3+	Cytochrome c1; Fe^2^+	1	0.22
Cytochrome c; Fe^3^+	Cytochrome c; Fe^2^+	1	0.25
Cytochrome a; Fe^3^+	Cytochrome a; Fe^2^+	1	0.29
1/2 O2 + H2O	H2O2	2	0.30
Cytochrome a3; Fe^3^+	Cytochrome a3; Fe^2^+	1	0.35
Ferricyanide	ferrocyanide	2	0.36
Cytochrome f; Fe^3^+	Cytochrome f; Fe^2^+	1	0.37
Nitrate	nitrite	1	0.42
Photosystem P700	.	.	0.43
Fe^3^+	Fe^2^+	1	0.77
1/2 O2 + 2H^+	H2O	2	0.816

The substrate oxidation mechanism by NAD⁺ involves concerted hydride transfer to one face of NAD⁺. Hydride transfer is possible since water is excluded from the active site. It is facilitated by removing a proton from an oxygen on an alcoholic substituent, for example, adjacent to the departing hydride. The negative charge on the oxide acts as a "source" of electrons, which can then flow through the hydride transfer to the positively charged ring nitrogen of NAD⁺, which acts as an electron "sink." This is crudely illustrated in the cartoon shown in Figure \(\PageIndex{7}\), which shows ethanol oxidation by the enzyme alcohol dehydrogenase.

Figure \(\PageIndex{7}\): Source and sink analogy for hydride transfer in alcohol dehydrogenase

For substrates like ethanol that lose a hydride from a methylene carbon atom that has two hydrogens, only one of them is lost (either the proR or proS) from the prochiral center.

The site on NAD⁺ that receives the hydride and the entire ring is planar with sp² hybridization. When bound to the enzyme, the hydride is transferred to the RE face of the ring. The same occurs in the reverse reaction when the hydride from NADH is transferred to the RE face of acetaldehyde. Using the Cahn-Ingold rules, RE and SI faces can be determined by prioritizing the substituents attached to the sp² carbon. The reversible transfer of the proR hydrogen to the Re faces of the reactants in the reversible conversion of ethanol to acetaldehyde is shown in Figure \(\PageIndex{8}\).

Figure \(\PageIndex{8}\): Reversible transfer of the proR hydrogen to the Re faces of the reactants in the alcohol dehydrogenase reaction

FAD has a more positive reduction potential than NAD⁺, so it is used for more "demanding" oxidation reactions, such as the dehydrogenation of a C-C bond to form an alkene. You will notice on standard reduction potential tables that the potential of FAD is often listed several times and depends on the enzyme. This is because the FAD is tightly bound to the enzyme. Hence, its tendency to acquire electrons depends on its environment, in much the same fashion as the pKa of an amino acid side chain (which reflects its tendency to release protons), which is affected by the environment of the amino acid side chain in the protein. The standard reduction potential for flavin enzymes ranges from -465 mV to +149 mV. Compare this to the free FAD/FADH² reduction potential, which in aqueous solution is -208 mV. The standard reduction potential of the flavin in D-amino acid oxidase, a flavoprotein, is about 0.0 V. Remember, the more positive the standard reduction potential, the more likely the reactant will be reduced and act as an oxidizing agent. Hence, the FAD in D-amino acid oxidase is a better oxidizing agent than free FAD. The K_D for binding of FAD to the enzyme is 10^-7 M compared to the K_D for binding of FADH₂, which is 10^-14 M. By gaining electrons, the flavin binds more tightly, which preferentially stabilizes the bound FADH₂ compared to the bound FAD. This shifts the equilibrium of FAD ↔ FADH₂ to the right, making the bound FAD a stronger oxidizing agent.

A mechanism for the 2-electron hydride reduction of FAD is shown in Figure 6 above.

Figure \(\PageIndex{9}\) below shows an interactive iCn3D model of D-amino acid oxidase bound to FAD and a trifluoroalanine (1C0L) .

Figure \(\PageIndex{9}\): D-amino acid oxidase bound to FAD and a trifluoroalanine (1C0L). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...wYfRAK9TBSt2c8

FAD is shown in spacefill and colored yellow. Only the amino acids interacting with FAD are shown as a surface with underlying CPK-colored sticks. The noncovalent interactions are shown as sticks. If you rotate the molecule, you will see that the FAD is almost completely buried and has extensive interactions with the protein, contributing to its low K_D.

Thermodynamically, FAD, especially when covalently or tightly bound (and not released) from an enzyme, has a greater drive to be reduced, making it a stronger oxidizing agent than NAD⁺.  Hence, it will be used biologically for more difficult oxidation reactions. Converting C-C to C=C is thermodynamically more difficult than converting C-OH to C=O.  This can likewise be inferred from standard reduction potentials (SRPs) for organic molecules.  The standard reduction potential (SRP) generally ranges from about +1 to +2.5V for the reduction of alkenes to alkanes.  For reducing an aldehyde to an alcohol, the SRP generally varies from about -2.5 to about 0V.  

Hence, reducing an alkene to an alkane is more favored than reducing an aldehyde to an alcohol. So, accordingly, the oxidation of an alkane to an alkene (reverse rx) is less favored than the oxidation of an alcohol to an aldehyde.  This makes some intuitive sense since the alcohol is already partially oxidized. So biologically, the tough oxidations use FAD as the oxidizing agent.  Of course, one of the toughest, the oxidation of H₂O to O₂ in photosynthesis, requires an even stronger oxidizing agent than O₂ (of course).  It is the oxygen-evolving complex, which we will see in the chapter on photosynthesis.

Can the standard reduction potential of a redox-active center in a protein be tuned by changing the environment of that center, much as the pKa of an acid side chain can by changing the polarity of the environment? The answer is yes. The active site of azurin, a cupredoxin, has a redox-active copper ion coordinated by a Cys and two His residues in a trigonal planar fashion. Met 121 serves as a weak axial ligand. Marshall et al. have reported a feasible method for manipulating the redox potential (Eo) of this active site. The wild-type azurin was mutated to alter its hydrophobicity and hydrogen-bonding capabilities while maintaining the overall architecture of the metal-binding site. Ser 46 was selected for mutation because it occupied a position similar to Asn in another cupredoxin, where it participates in an important H-bond between two ligand-binding loops. An N47S mutation, which strengthened the hydrogen bond between the two ligand-containing loops, increased Eo by ~130 mV while preserving metal binding site architecture as determined by UV-Vis spectroscopy. They also compared a M121Q mutant with wild-type M121 and with a M121L mutant. A plot of E^o vs. log partition coefficient for the transfer of the side chain from water to octanol was essentially linear, with a positive slope, indicating that the standard reduction potential depended on the hydrophobicity of the weakly coordinating ligand in the metal-binding region. This behavior extended to double mutants (where one set of mutants involved M121). The investigators were able to tune the E^o over a 700 mV range!

Monooxygenases

An example of monooxygenases is the hydroxylases, which hydroxylate amino acids such as tryptophan and tyrosine to form 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine (dopa), respectively. These two substances can be decarboxylated using PLP-dependent enzymes to form the neurotransmitters 5-hydroxytryptamine (5-HT or serotonin) and dopamine. The latter can be hydroxylated again to form norepinephrine, which is subsequently methylated to form epinephrine. LSD and amphetamine are analogs of serotonin and dopamine, respectively. The derivatives of tryptophan and tyrosine are shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Derivative of tryptophan and tyrosine derived from monooxygenases

Since these monooxygenases use dioxygen, you might expect the enzymes to use the motifs described in the previous section to facilitate their reaction with dioxygen. The enzyme contains a metal ion (Fe²⁺) bound to a heme in the protein. In addition, the dioxygen reduction products that are eventually used to hydroxylate the substrate remain bound to the enzyme.

Tyrosine 3-monooxygenase (Tyrosine hydroxylase)

Tyrosine hydroxylase is a homotetramer that uses an Fe²⁺ ion and a biopterin cofactor for hydroxylation. Dihydroxyphenylalanine (DOPA) biosynthesis is the rate-limiting step in catecholamine synthesis.

Figure \(\PageIndex{11}\) shows a possible mechanism for the rat enzyme.

Figure \(\PageIndex{11}\): A possible mechanism for rat tyrosine hydroxylase, a monooxygenase. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/134/

The Fe²⁺ ion binds O_2. Some mechanisms involve a single-electron transfer from the pterin ring to the bound O₂, forming Fe²⁺-O₂^-(bound superoxide) and a pterin radical cation. This is unstable and forms Fe²⁺-μ-peroxypterin, followed by heterolytic cleavage of the peroxo O-O bond. Ultimately, hydroxpterin and an Fe⁴⁺O oxospecies form, which then hydroxylates tyrosine.

Figure \(\PageIndex{12}\) below shows an interactive iCn3D model of rat tyrosine hydroxylase with bound cofactor analog and iron (2TOH).

Figure \(\PageIndex{12}\): Rat tyrosine hydroxylase with bound dihydrobiopterin analogue and iron (2TOH). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...KeKiLDfmeNFfr7

Four monomers in the homotetramer are shown. The cofactor analog (HBI), 7,8-dihydrobiopterin, is shown in spacefill. Phenylalanine 300 has been self-hydroxylated by the enzyme to produce meta-tyrosine (MTY300), which, along with the other key amino acids in the active site, is labeled in CPK-colored sticks in the gray monomer. The pterin ring interacts with Phe 300 via pi stacking, facilitating self-hydroxylation. The Fe ion is far (5.6 Å) from the carbon on pterin. This suggests that O₂ might bridge the reacting pterin C- and the Fe ion in a Fe²⁺-μ-peroxypterin complex.

Tryptophan hydroxylase

This enzyme also uses a tetrahydropterin cofactor and an Fe ion to form the hydroxylating intermediates. As in the case of tyrosine hydroxylase, both the amino acid substrate and the cofactor must be present for oxygen to be activated. Otherwise, it only oxidizes Fe²⁺ to Fe³⁺. This serves as a protective mechanism, preventing unnecessary activation of O₂ and avoiding the formation of soluble ROS.

Figure \(\PageIndex{13}\) shows a possible mechanism without catalytic residues for tryptophan hydroxylase.

 

Figure \(\PageIndex{13}\): possible mechanism without catalytic residues for tryptophan hydroxylase, adapted from Kenneth M. Roberts, Paul F. Fitzpatrick, https://doi.org/10.1002/iub.1144

Cytochrome P450s

Cytochrome P450 (CYP) consists of a large group of monooxygenases that contain a heme that absorbs maximally at 450 nm. They catalyze the following reaction:

RH + NAD(P)H + H⁺ + O₂ → ROH + NAD(P)⁺ + H₂O

An example is cytochrome P450cam, which hydroxylates camphor, a large, completely nonpolar aromatic molecule. The enzyme has been called the "biological equivalent of a blowtorch," as it can, at room temperature, stereospecifically hydroxylate nonactivated hydrocarbons at physiological temperatures. This reaction proceeds without stereospecificity only at high temperatures, in the absence of a catalyst. Remember from the previous chapter section that dioxygen is a ground-state triplet radical that is not reactive with carbon atoms (which are singlets) unless converted to a singlet state, a process that requires significant energy. Cytochrome P450s get around this problem by binding to an Fe ion in the heme to form common intermediates such as oxos, oxides, and peroxides. The molecular orbitals of the bound dioxygen are "singlet-like."

Remember from introductory chemistry that transition-metal ligands are named according to a specific nomenclature. Some are shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Common oxygen transition metal ligands

In their names, η is the hapticity (the number of atoms of a ligand attached to a metal), and µ is the number of metal atoms bridged by a ligand. For Fe ions and oxygen ligands, some examples include Fe^III–O₂⁻ (superoxo) and Fe^IV=O (ferryl-oxo)

If the aromatic substrate gets oxidized, something must be reduced. That something is, of course, O₂. Electrons for the reduction of O₂ come from the oxidized substrate and the input of electrons through NAD(P)H. Cytochrome P450s are microsomal proteins, and most require another protein, NADPH-cytochrome P450 reductase (CPR). Before we look at the structure and mechanism of cytochrome P450, let's look at this microsomal protein first. CPR catalyzes this reaction:

NADPH + oxidized CytoP450 (Fe³⁺-heme b) + H⁺ ↔ NADP⁺ + Oxidized CytoP450 (Fe²⁺-heme b)

The protein contains multiple domains and results from a fusion of genes from flavodoxin (which binds FMN) and FAD reductases. In addition, it contains an NADP-binding domain. It ultimately accepts a hydride ion (2 electrons) from NADPH and then transfers electrons to FMN (in one-electron steps). These are used to reduce the heme, which then activates dioxygen (oxidation number of 0) for hydroxylation reactions by cytochrome P450. In the hydroxylated organic product and water, oxygen has an oxidation number of -2.

Figure \(\PageIndex{15}\) below shows an interactive iCn3D model of rat NADPH-cytochrome P450 reductase (1AMO).

Figure \(\PageIndex{15}\): Rat NADPH-cytochrome P450 reductase (1AMO). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...nMh9ByRbsWk4B8

NADP⁺ is labeled as NAP. The enzyme is unique because it has binding sites for FAD and FMN (similar to nitric-oxide synthase). The flavins are aligned for electron transfer. The cleft in the enzyme presumably binds cytochrome P450. The protein has short (20 amino acids) lumenal sections, followed by a 20 amino acid membrane-spanning helix (not shown in the above structure), followed by a large cytosolic domain, where it can interact with cytochrome P450, which is found in multiple places, including the ER, microsomal, and mitochondrial membranes, as well as the cytoplasm.

The cytochromes P450s contain a heme, which, instead of reversibly carrying dioxygen (as in myoglobin and hemoglobin), activates dioxygen for hydroxylation reactions involving aromatic, nonpolar substrates. Hydroxylation of these substrates increases their solubility, facilitating their elimination from the body. Epoxide intermediates and products are often produced, which can open up through nucleophilic attack using an alcohol (sugar derivative), as illustrated in Figure \(\PageIndex{16}\).

Figure \(\PageIndex{16}\): Cytochrome P450 hydroxylation reaction to increase target solubility

This type of reaction also converts steroids to different biologically active ones.

The hydroxylation reaction can also lead to reactions with amines, including those on nucleotide bases in DNA, resulting in the formation of large adducts, as illustrated in Figure \(\PageIndex{17}\).

Figure \(\PageIndex{17}\): Cytochrome P450-mediated formation of carcinogens.

Hence, cytochrome P450 can activate aromatic substrates to become carcinogens.

The cytochrome P450 family of genes/proteins is inducible on exposure to nonpolar aromatic molecules such as dioxin. These nonpolar molecules can enter the cytoplasm and bind to the aryl hydrocarbon receptor (AhR), which is bound to the heat shock protein Hsp90. Upon dioxin binding (for example, TCDD), the AhR-TCDD complex dissociates from Hsp90 and migrates to the nucleus, where it binds a protein called Amt. The AhR-Amt complex is an enhancer/transcription factor that facilitates transcription of cytochrome P450 genes.

Figure \(\PageIndex{18}\) illustrates the activation of cytochrome P450 gene expression on exposure to nonpolar aromatic molecules such as dioxin.

Figure \(\PageIndex{18}\): Activation of cytochrome P450 and other gene expression on exposure to nonpolar aromatic molecules such as dioxin

On binding to a ligand, the AhR is activated and enters the nucleus, where it binds to ARNT on the aryl hydrocarbon response element (AhRE) and promotes transcription of downstream genes, including cytochrome P450 family 1 subfamily A member 1 (CYP1A1) and interleukin-1 (IL-1). CYP1A1 is in the CYP1A family and promotes the activation of procarcinogens and the hydroxylation of steroid hormones like estrogens. It also participates in the metabolism of steroidal hormones, including estrogens. ARNT is the aryl hydrocarbon receptor nuclear translocator; AhRE is the aryl hydrocarbon response element; CYP1A1, XAP2 is the aryl hydrocarbon receptor interacting protein; AHRR is the aryl-hydrocarbon receptor repressor; IL-11 is interleukin 17; Hsp90 is heat shock protein 90, and p23 is prostaglandin E synthase 3

Dioxin has been shown to affect estrogen-mediated activities. Estrogens, small hydrophobic hormones derived from cholesterol, enter cells and bind to cytoplasmic estrogen receptors, which then dimerize and bind to the estrogen response element (ERE), initiating transcription. Tamoxifen, a drug derived from the yew plant, blocks the biological effects of the estrogen receptor. Although it binds to the estrogen receptor, it doesn't elicit the same conformational changes in the protein, preventing the bound receptor from binding to the estrogen response element and recruiting the other proteins needed for estrogen-dependent gene transcription. It is used in chemotherapy and the prevention of estrogen-dependent breast cancer cells.

How does dioxin interfere with estrogen signaling? Ahr and ARnt contain a basic helix-loop-helix motif, which mediates their interaction with DNA. Upon complex formation, detoxification genes are activated. The dioxin-Ahr-Arnt complex can also bind to the estrogen receptor, activating genes containing an estrogen response element (ERE) in the absence of estrogen. However, if estrogen is present, ERE-mediated gene expression inhibition is observed. Dioxins can be potent dysregulators of estrogen-induced gene expression. Such changes in estrogen activity could help explain the pro- and inhibitory effects of dioxin on estrogen-mediated cellular responses, as well as its possible effects on the immune system and cancer development.

Given the importance of the cytochrome P450s, we'll offer two variant portrayals of their mechanism. Figure \(\PageIndex{19}\) shows the overall catalytic cycle of the enzyme with associated redox changes in the generic substrate, RH, and the Fe heme ion.

Figure \(\PageIndex{19}\): Catalytic cycle of cytochrome P450 with the generic substrate RH and the Fe heme ion

The key hydroxylating agent appears to be the formal FeO³⁺ species, shown in step 7.

A possible mechanism for the hydroxylation of estrone by cytochrome P450 from Bacillus megaterium is shown below in Figure \(\PageIndex{20}\). It follows the general catalytic cycle shown above.

Estrone + NADH + H⁺ + O₂ → 2-hydroxyestrone + NAD⁺ + H₂O

Figure \(\PageIndex{20}\): Possible mechanism for the hydroxylation of estrone by cytochrome P450 from Bacillus megaterium after https://www.ebi.ac.uk/thornton-srv/m...csa/entry/699/

As with the previous generic mechanism, the two added electrons derive from NADH, which passes a hydride to FAD, which then passes single electrons to FMN, which passes them onto the heme. The exact species involved in several parts of the mechanism are not entirely clear. For instance, a Fe⁴⁺-oxo complex has been proposed.

Let's show the structure of a human cytochrome P450 1A1 active in drug metabolism and the activation of benzo[a]pyrene (a fused benzene and α-pyrone ring), a component of cigarette smoke, into a carcinogen. Figure \(\PageIndex{21}\) below shows an interactive iCn3D model of human microsomal (ER) cytochrome P450 2A6 complexed with coumarin (1Z10).  Coumarin is a benzo-α-pyrone found in many plants, like tonka beans and some types of cinnamon.  It has a sweet, vanilla-like scent and bitter taste.  

Figure \(\PageIndex{21}\): Human microsomal cytochrome P450 2A6 complexed with coumarin (1Z10). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...J8LxFpEyGdeab6

The heme (Heme) and coumarin (COU) are shown in spacefill and CPK colors, with labels.  The amino acids lining the coumarin-binding pocket are shown in CPK-colored sticks, unlabeled except for Asn 297.  The coumarin is bound in a completely nonpolar site but forms one hydrogen bond with Asn 297 (labeled), which helps position it.  

Dioxygenases

An example of a dioxygenase is the cyclooxygenase activity of prostaglandin synthase. This enzyme, often called cyclooxygenase (COX), is an integral membrane protein found in the ER membrane and is a homodimer (with two hemes). It catalyzes two different reactions. One is the addition of two dioxygen molecules to arachidonic acid - 20:4^Δ5, 8, 11, 15 (which is liberated from the C2 position of phospholipid membranes by phospholipase A2 upon appropriate signaling) to form prostaglandin PGG2. This molecule, with 5 chiral centers, arises from arachidonic acid, which has one. The cyclooxygenase activity is buried in the membrane, from where the arachidonic acid can readily access its active site. The active site is at the end of a hydrophobic channel (the arachidonic acid binding site) and extends from the membrane-binding region to a buried heme. PGG2 can be further metabolized to PGH2 by the addition of two electrons via the enzyme's hydroperoxidase activity, located at the other end of the enzyme. This activity converts the peroxide functional group in PGG2 into an alcohol. There is one heme per monomer, which acts in both the cyclooxygenase and peroxidase activities. Each monomer of the dimer has both enzymatic activities. A possible abbreviated reaction mechanism is shown in Figure \(\PageIndex{22}\).

 

Figure \(\PageIndex{22}\): A possible abbreviated reaction mechanism for dioxygenase cyclooxygenase.

In summary:

• The carboxylate of arachidonic acid is coordinated to Arg 120 and Tyr 355.
• The C13 pro(S) H atom of arachidonic acid is close to Tyr 385, which allows its abstraction.
• This results in a radical centered on C11 that reacts with dioxygen to form a peroxyl radical.
• Attack by dioxygen at C11 occurs from the side of the substrate opposite to that of hydrogen abstraction.
• The oxygen radical at C11 cyclizes by attacking C9.

The C13 proS hydrogen atom (not proton) is removed from bound arachidonic acid by a free radical form of Tyr 385, which acts as an oxidizing agent. A site-specific mutant in which Phe replaces Tyr 385 is inactive. How is the Tyr free radical formed? Based on the single-electron standard reduction potential (0.9 V for Tyr and -0.2 to + 0.2 V for Fe³⁺ in the bound heme), it appears that the heme iron is not a potent enough oxidizing agent to accomplish this task. However, oxygen bound to the heme iron could be converted to a peroxide, forming an Fe⁴⁺-oxo complex (which has also been proposed for cytochrome P450). The Fe⁴⁺ ion is a more potent oxidizing agent (standard reduction potential of approximately 1 V, sufficient for oxidation of Tyr 385. Another possibility is that the peroxide activator (in forming the ferryl-oxo ligand) is NO (nitric oxide, a free radical). NO is produced by immune cells (such as macrophages) during immune activation. The NO might react with superoxide (also a radical, possibly formed during an oxidative burst in macrophages during immune stimulation) to form peroxynitrite (NO^3-). This can donate an oxo group to Fe³⁺, forming the Fe⁴⁺-oxo complex, which could then oxidize Tyr to the free radical form. There might be other mechanisms as well to generate the Tyr free radical, since just adding organic peroxides to the enzyme will generate it. After abstraction of the proS H atoms, a carbon-centered free radical at C11 results, which reacts with oxygen, as shown below. The exact form of oxygen that reacts is unclear, but it is presumably either a peroxy or an activated singlet form.

Oxidases

This class of enzymes does not incorporate dioxygen into an organic substrate. Rather, it accepts electrons released from an organic substrate via intermediate electron carriers (such as ubiquinone and cytochrome C) to form superoxide (as in NADPH oxidase), hydrogen peroxide (as in xanthine oxidase), or water (as in cytochrome c oxidase). The mechanism of cytochrome C oxidase again supports our expectations about enzymes that use dioxygen. Dioxygen binds to metals in the enzyme. One oxygen atom binds to the heme Fe²⁺ of cytochrome a3, which is bound to the enzyme, while the other binds to a Cu¹⁺ of Cu B. All oxygen reduction intermediates remain bound to the enzyme. Four electrons are added from four different cytochrome C molecules, which serve as mobile carriers of electrons.

We will explore cytochrome C oxidase in detail in Chapter 19.1: Electron-Transfer Reactions in Mitochondria. Figure \(\PageIndex{23}\) shows cartoon versions of several oxidases.

Figure \(\PageIndex{23}\): Examples of oxidases

Another example of an oxidase is monoamine oxidase. Mitochondrial monoamine oxidase catalyzes the oxidative deamination of certain neurotransmitters after postsynaptic neurons have taken them up during inactivation. The reaction is shown in Figure \(\PageIndex{24}\).

Figure \(\PageIndex{24}\): Monoamine oxidase reaction

A Schiff base is formed, which is then hydrolyzed, incorporating unlabeled oxygen into the oxidized molecule.

Biological Oxidations: Methane to CO₂

In the previous chapter section, we discussed the progressive oxidation of methane by 2 electron losses to form methanol, formaldehyde, formic acid, and CO₂, with a progressive increase in oxidation number for the C by +2 (from -4 in methane to +4 in CO₂), as reviewed in Figure \(\PageIndex{25}\).

Figure \(\PageIndex{25}\):: Progressive stages in the oxidation of methane

Methanotrophs are aerobic bacteria that use methane as an energy source, converting it in a series of two-electron oxidations, as shown above, to carbon dioxide. The enzymes involved in this sequential process are methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. Methane monooxygenase exists in a soluble and membrane form, both of which are part of a larger complex. Both have a hydroxylase (which uses dioxygen to add O to methane), and the membrane form has recently been shown to be associated with methanol dehydrogenase in a larger complex consisting of trimers of each enzyme (the hydroxylase and the dehydrogenase).

Heme Proteins

So far in this course, we have examined three different kinds of heme proteins.

• The first is that hemoglobin and myoglobin serve as carriers of dioxygen. Even though they bind one of the best oxidizing agents around (dioxygen), the heme Fe²⁺ is not oxidized to Fe³⁺. If it does, as in the case of met-Hb, the protein loses its ability to carry oxygen.

• Cytochrome C, conversely, does not bind dioxygen but serves as a carrier of electrons, which are passed to dioxygen in Cytochrome C oxidase. As an electron carrier, its Fe ion readily cycles between the 2+ and 3+ states.
• Finally, the Fe²⁺ in the heme of the cytochrome P450s (so named since they have an absorbance maximum at 450 nm when they bind CO) does both. It binds dioxygen and cycles between the 2+ and 3+ states, activating dioxygen for hydroxylation reactions.

The heme and amino acid ligands' structure and their absorbance spectra are shown in Table \(\PageIndex{3}\) below.

hemoglobin
cytochrome C
Cytochrome P450

P450: Fujishiro et al. J Biol Chem. 2011 Aug 26; 286(34): 29941–29950. Published online 2011 Jun 30. doi: 10.1074/jbc.M111.245225. CC BY license.

Cyto C: Hulko et al. December 2011. Sensors 11(6):5968-80. DOI:10.3390/s110605968. Creative Commons Attribution 3.0 Unported

Hemoglobin: Nitzan et al. July 2014, Medical Devices: Evidence and Research 7(1):231-9. DOI:10.2147/MDER.S47319. Creative Commons Attribution-NonCommercial 3.0 Unported

How could heme serve such diverse functions? We can explain this by referring to one of the course's main themes - structure mediates function. The environment of each heme must be different. The protein ligands coordinating the Fe ions are different. The 5th ligand is the proximal His in hemoglobin, while dioxygen binds to the 6th site. The 5th and 6th ligands in cytochrome C are His and Met, respectively. In cytochrome P450, the 5th site is occupied by Cys, and the 6th by dioxygen. Presumably, the environments surrounding the hemes are different as well. Once again, we have seen an analogous example in which the microenvironment influences chemical properties. The pKa of a given amino acid side chain can vary considerably depending on the polarity of the local environment. Likewise, the standard reduction potential of tightly bound FAD/FADH₂ depends on the microenvironment.

As we have seen (from studies of heme proteins and cellular oxidative enzymes), transition metals such as Fe, Zn, and Cu play vital biological roles as binding sites and cofactors in many reactions. Yet they also pose problems since they can lead to cellular oxidative damage. As we saw with cytoplasmic metallothioneins, which bind to heavy metals and protect the cell from such damage, many proteins are involved in binding and regulating transition metals in the cell. Integral membrane proteins are required to bind and transport these cations into the cytoplasm. Other proteins act as sensors of transition-metal concentration (such as latent transcription factors, which bind heavy metals and become active transcription factors for metallothioneins). Others act as chaperone proteins, binding metal ions and transferring them to apometalloproteins.  Recent work suggests that transporters and chaperones are involved in metal-ion biology and bind these ions with unusual coordination geometries, presumably facilitating their transfer to the apo-target protein.

The transition metals Zn and Fe are often found in E. coli at 0.1 mM, whereas Cu and Mn are present at 10-100 μM. Also, about one-third of all proteins bind metal ions and are classified as metalloproteins. Mass balance suggests that metal ions would be distributed among proteins with low, intermediate, and high metal-binding affinity, as well as in free pools, which can be toxic to cells. Depending on their Kd for metal ion binding, metalloproteins would be in various states of ligation. The free concentration of some ions (Cu and Zn) is so low that newly synthesized apoproteins that bind these ions would not obtain the ions from the free pool. In such cases, metal chaperones would be required.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter provides a unified framework for understanding biological oxidation reactions, organizing the diverse enzymes involved by the identity of their oxidizing agent and the fate of dioxygen.

The chapter begins by contrasting the chemical oxidizing agents used in laboratory settings — permanganate and chromate — with those used in living cells. Most biological oxidations occurring in central metabolic pathways rely on NAD⁺ or FAD rather than dioxygen itself. NAD⁺ operates exclusively through concerted two-electron hydride transfer, accepting a hydride from the substrate at one specific face of its nicotinamide ring with the assistance of an active-site base. This strictly two-electron mechanism makes NAD⁺ incompatible with dioxygen, which reacts via single-electron steps. FAD, by contrast, can participate in both one- and two-electron transfers and is therefore suited to more demanding oxidations, including the dehydrogenation of C–C bonds to form alkenes. Because FADH₂ is susceptible to uncontrolled reaction with cellular dioxygen, FAD is held tightly within its enzyme — with dissociation constants as low as 10⁻¹¹ M — ensuring that only the appropriate oxidizing agent accesses the reduced flavin. The standard reduction potential of enzyme-bound FAD varies over a remarkable 600+ mV range depending on the local protein environment, a tunability directly analogous to the influence of protein microenvironment on amino acid pKa values.

Enzymes that use dioxygen as a substrate are classified by how many oxygen atoms are incorporated into the organic product. Monooxygenases insert one oxygen atom into the substrate while reducing the other to water. The pterin- and Fe-dependent tyrosine and tryptophan hydroxylases illustrate this class, converting these amino acids into precursors for the catecholamine and indolamine neurotransmitters. The cytochrome P450 superfamily represents the most versatile and consequential monooxygenases in biology, capable of stereospecifically hydroxylating chemically inert, nonpolar hydrocarbons at physiological temperatures — a reaction that would require high temperatures without a catalyst. Their catalytic cycle centers on a heme iron that cycles between Fe²⁺ and Fe³⁺ states, binds and activates dioxygen, and generates a reactive Fe⁴⁺-oxo (ferryl) intermediate that performs the hydroxylation. NADPH donates electrons via a dedicated reductase containing both FAD and FMN, which relay single electrons to the heme. Hydroxylation of nonpolar aromatics increases their aqueous solubility, facilitating excretion — a detoxification function. However, the same chemistry can convert polycyclic aromatic hydrocarbons such as benzo[a]pyrene into reactive epoxides that form adducts with DNA bases, explaining how cytochrome P450 induction by dioxin and cigarette smoke components can paradoxically promote carcinogenesis. Dioxin induces P450 expression by binding the aryl hydrocarbon receptor (AhR), which then enters the nucleus, partners with ARNT, and drives transcription of CYP genes; this pathway also dysregulates estrogen receptor signaling. Dioxygenases, exemplified by prostaglandin synthase/cyclooxygenase (COX), incorporate both atoms of dioxygen into the substrate. COX converts arachidonic acid into PGG2 through a radical mechanism initiated by a tyrosine free radical at the active site, ultimately generating a bicyclic peroxide with five new stereocenters. A separate peroxidase activity within the same enzyme then reduces PGG2 to PGH2.

Oxidases represent a third class: they accept electrons from organic substrates through intermediate carriers and deliver them to dioxygen, but do not incorporate oxygen atoms into the organic product. Depending on how many electrons are transferred, the dioxygen is reduced to superoxide (one electron, as in NADPH oxidase), hydrogen peroxide (two electrons, as in xanthine oxidase), or water (four electrons, as in cytochrome C oxidase). The chapter closes by comparing heme function across three protein families — hemoglobin/myoglobin (O₂ transport without iron oxidation), cytochrome C (electron shuttling between Fe²⁺ and Fe³⁺), and cytochrome P450 (O₂ binding and activation for hydroxylation) — illustrating how the same prosthetic group achieves radically different chemistry through differences in axial ligands and protein microenvironment, a recurring theme throughout the text.