The Role of Tetrahydrobiopterin in Nitric Oxide Synthase

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Nitric oxide ( NO) was discovered as the endothelium-derived relaxation factor. It is produced in the central nervous system, lungs, macrophages, and vascular endothelial cells in response to a variety of stimuli. It initiates a cGMP signaling cascade.

In smooth muscle, nitric oxide activates a cytosolic guanylate cyclase by reacting with a heme-containing regulatory subunit and causing a conformational change. This results in smooth muscle relaxation and hence vasodilatation as a result of reduced intracellular calcium in response to the increased concentration of cGMP. This role of nitric oxide in stimulating guanyl cyclase explains the clinically useful vasodilatory action of nitroglycerine and aryl nitrites, which decompose in vivo to yield nitric oxide. Endothelium-derived nitric oxide also inhibits platelet aggregation, again acting byway of activation of guanylate cyclase.

In the central nervous system, nitric oxide formation from arginine is the immediate response of the glutamate-activated N-methyl-D-aspartate (NMDA) receptors. Again, it acts intercellularly, being released from neurons and acting on neighboring glial cells by stimulation of guanylate cyclase. These actions of nitric oxide are transient; activated macrophages continue to produce nitric oxide from arginine, in considerably higher concentrations, as a part of their cytotoxic action.

As shown in Figure 10.11, the reaction catalyzed by nitric oxide synthase is hydroxylation of arginine; N-hydroxy-arginine then decomposes to citrulline and nitric oxide. It is unclear whether the immediate product of the enzyme is nitric oxide itself or the nitroxyl anion NO-; the addition of superoxide dis-mutase in vitro increases the formation of nitric oxide, but this could be the

Amide Anion

arginine Nm-hydroxyarginine

Figure 10.11. Reaction of nitric oxide synthase (EC

arginine Nm-hydroxyarginine

Figure 10.11. Reaction of nitric oxide synthase (EC

result of either oxidation of nitroxyl to nitric oxide or the removal of superoxide, which reacts with nitric oxide to yield peroxynitrite (Alderton et al., 2001).

Although the enzyme requires tetrahydrobiopterin for activity, the role of pterin is not clear. Nitric oxide synthase is a heme-containing enzyme that reacts with a reduced flavin; by analogy with cytochrome P450-dependent enzymes, it could catalyze the hydroxylation of arginine without requiring a pterin cofactor. There is no evidence of cycling between tetrahydrobiopterin and dihydrobiopterin, as in the case of the aromatic amino acid hydroxy-lases (Section 10.4.1), although there is evidence of formation of the trihydro-biopterin radical as an intermediate in the reduction of heme. In addition to a possible direct role in hydroxylation of arginine, tetrahydrobiopterin may also act in the following ways:

1. to inhibit the formation of superoxide and hydrogen peroxide;

2. to promote formation of, and stabilize, the active dimer (the monomer of the enzyme is inactive);

3. to be an allosteric modifier of either the arginine binding site or the environment of the reactive heme group; and

4. to protect the enzyme against autoinactivation.


Three human redox enzymes, and a variety of bacterial enzymes, contain molybdenum chelated by two sulfur atoms in a modified pterin: molybdopterin (see Figure 10.1). In sulfite oxidase, the other two chelation sites of the molybdenum are occupied by oxygen; in xanthine oxidase/dehydrogenase (Section 7.3.7) and aldehyde oxidase, one site is occupied by oxygen and one by sulfur. In some bacterial enzymes, molybdopterin occurs as a guanine dinu-cleotide rather than free. In others, tungsten rather than molybdopterin is the chelated metal; there is no evidence that any mammalian enzymes contain tungsten.

Unusually for redox reactions involving metal ions, the molybdenum undergoes a two-electron reaction, cycling between MoVI and IV; however, the enzymes also have a reactive heme and/or an iron-sulfur cluster (which is a single-electron acceptor/donor). Thus, there must be intermediate formation of Mo^ (Kisker et al., 1997; Rajagopalan, 1997; Nishino and Okamoto, 2000).

A very small number of children have been reported who are unable to synthesize molybdopterin; they show severe neurological abnormalities shortly after birth and fail to survive more than a few days. As expected from the metabolic roles of molybdopterin, they have low blood concentrations of uric acid and sulfate, and abnormally high levels of xanthine and sulfite. The neurological damage is probably caused by sulfite, because similar abnormalities are seen in children with isolated sulfite oxidase deficiency (Reiss, 2000).


The structure of vitamin B12 is shown in Figure 10.12. The corrin ring is a tetrapyrrole with fused A to D rings; the term corrinoid is used as a generic descriptor for cobalt-containing compounds of this general structure, which, depending on the substituents in the pyrrole rings, may or may not have vitamin activity. Cobalamins are corrinoids that have a dimethylbenzimidazole nucleotide attached to the D ring and chelating the central cobalt atom. The term vitamin B12 is used as a generic descriptor for the cobalamins - those corrinoids having the biological activity of the vitamin.

A number of noncobalamin corrinoids have activity in microbiological assays and thus appear to be vitamin B12, although they have no vitamin activity and may have antimetabolite activity. The common method of measuring vitamin B12 and that required by law in some countries (including the United States) is a microbiological growth assay using Lactobacillus leichmanii, for which a number of noncobalamin corrinoids are growth factors. As a result, a number of foods that contain only noncobalamin corrinoids are nonetheless lawfully described as containing vitamin B12 (Herbert, 1988).

As shown in Figure 10.12, four of the six chelation sites of the cobalt atom of cobalamin are occupied by the nitrogens of the corrin ring and one by the nitrogen of the dimethylbenzimidazole side chain. The sixth site may be occupied by the following ligands in biologically active vitamers:

• OH- (hydroxocobalamin) or H2O (aquocobalamin), depending onpH

• 5'-deoxy-5'adenosine (adenosylcobalamin)


Figure 10.12. Vitamin B12. Four coordination sites on the central cobalt atom are occupied by the nitrogen atoms of the corrin ring, and one by the nitrogen of the dimethyl-benzimidazole nucleotide. The sixth coordination site may be occupied by: CN- cya-nocobalamin, Mr = 1355.4; OH - hydroxocobalamin,Mr = 1346.4; H2O aquocobalamin, Mr = 1347.4; -CH3 methylcobalamin, Mr = 1344.4; and 5'-deoxyadenosine adenosyl-cobalamin, Mr = 1579.6.


Figure 10.12. Vitamin B12. Four coordination sites on the central cobalt atom are occupied by the nitrogen atoms of the corrin ring, and one by the nitrogen of the dimethyl-benzimidazole nucleotide. The sixth coordination site may be occupied by: CN- cya-nocobalamin, Mr = 1355.4; OH - hydroxocobalamin,Mr = 1346.4; H2O aquocobalamin, Mr = 1347.4; -CH3 methylcobalamin, Mr = 1344.4; and 5'-deoxyadenosine adenosyl-cobalamin, Mr = 1579.6.

Sulfitocobalamin, with a sulfite ligand, occurs in some foods as a result of processing, but is poorly absorbed.

The cobalt atom is in the Co3+ oxidation state in hydroxo-, aquo-, methyl-, and cyanocobalamins; in the Co+ oxidation state in adenosylcobalamin; and, transiently, in the demethylated prosthetic group of methionine synthetase (Section 10.8.1).

Although cyanocobalamin was the first form in which vitamin B12 was isolated, it is not an important naturally occurring vitamer, but rather an artifact caused by the presence of cyanide in the charcoal used in the extraction procedure. It is more stable to light than the other vitamers, and is commonly used in pharmaceutical preparations. Photolysis of cyanocobalamin in solution leads to the formation of aquocobalamin or hydroxocobalamin, depending on pH.

Hydroxocobalamin is also used in pharmaceutical preparations and is better retained after parenteral administration than is cyanocobalamin.

Small amounts of cyanocobalamin are found in the bloodstream (about 2% of total plasma vitamin B12) apparently as part of the metabolism of cyanide derived from food (and tobacco smoke), but not in erythrocytes or tissues. If it is not converted to aquo- or hydroxocobalamin, cyanocobalamin may have antivitamin action and has been implicated in the neurological damage associated with chronic cyanide intoxication seeninparts ofwest Africa, where the dietary staple, cassava, is rich in cyanogenic glycosides.

The major plasma vitamer is methylcobalamin, accounting for 60% to 80% of plasma vitamin B12, withupto 20% as adenosylcobalamin and the remainder mainlyhydroxocobalamin. In tissues, the major vitamer is adenosylcobalamin (about 70% in liver), with about 25% as hydroxocobalamin and less than 5% as methylcobalamin.


Very small amounts of vitamin B12 can be absorbed by diffusion across the intestinal mucosa. But, under normal conditions, this is insignificant, accounting for less than 1% of large oral doses. The major route of vitamin B12 absorption is by way of attachment to a specific binding protein in the intestinal lumen.

This binding protein is intrinsic factor, so-called because in the early studies of pernicious anemia (Section 10.9.2), it was found that two factors were required- an extrinsic or dietary factor, which we now know to be vitamin B12, and an intrinsic or endogenously produced factor. Intrinsic factor is a glycoprotein (Mr 44,000, containing 15% carbohydrate) secreted by the gastric parietal cells, which also secrete hydrochloric acid. The secretion of both intrinsic factor and gastric acid is stimulated by vagus nerve stimulation, histamine, gastrin, and insulin.

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