Pterin oxidation states

Stronger chemical evidence for the presence of a dithiolene in molybdopterin was obtained when the mild alkylation reagent iodoacetamide effectively trapped the dithiolene (65). This reaction yielded a derivative whose characterization by FAB mass spectrometry and nuclear magnetic resonance (NMR) was consistent with the structure shown in Fig. 5. The method appeared to leave the side chain intact and preserved the pterin oxidation state. From this experiment the view persisted that molybdopterin is a disubstituted dithiolene bearing a reduced pterin and a short chain terminated with a phosphate. 

The presence of a pterin moiety in mononuclear Mo enzymes has stimulated work in this area. Concensus has replaced earlier confusion and controversy concerning the formulations, metal and pterin oxidation states, and electronic descriptions of Mo—pterin complexes. High-valent, monomeric oxo—Mo pterin complexes are diamagnetic yet they are formulated, on the basis of X-ray photoelectron and NMR spectroscopic data, as Mov species This formulation and (partial) representations like (265) and (266) attempt to reflect the extent of electron pair delocalization in these non-innocent (buffered588) systems, not the existence of metal- and pterin-based diradicals.630... 

Pterins — These are pigments derived from pteridine skeletons. All natural pterins are 2-amino-4-hydroxypteridines bearing various substituents at Cg and C7 and having different oxidation states of N5 and Ng. 

Tetrahydrofolate (THF, 6) is a coenzyme that can transfer Cj residues in different oxidation states. THF arises from the vitamin folic acid (see p. 366) by double hydrogenation of the heterocyclic pterin ring. The Ci units being transferred are bound to N-5, N-10, or both nitrogen atoms. The most important derivatives are ... 

The importance of the dihydro and tetrahydro oxidation states of pterins in biology has stimulated interest in the study of the chemical properties of these compounds, especially with respect to electron-transfer and radical reactions. It has become apparent, perhaps unsurprisingly, that the stability and reactivity of these oxidation states are very sensitive to substituent effects and the much greater stability of the fully conjugated pteridines is most evident. The oxidation of tetrahydropterins and the reduction of dihydropterins have become especially important in the chemistry of nitric oxide production in nature and in oxidative stress but the accumulation of relevant facts has not led so far to a detailed understanding of the chemical property relationships. Relevant information is summarized in the following section. 

Naturally occurring pterin derivatives have existed in 3 oxidation states pterin (4 aromatic), dihydropterin (e.g., 5-8) and tetrahydropterin (9). In the present review, we do not refer to reduced pterin derivatives with reduced pyrimidine structures. The reduced pterin derivatives, dihydropterin and tetrahydropterin are readily oxidized to the corresponding aromatic form (4) under aerobic conditions. Based on the location to which hydrogen atoms are added, 4 kinds of dihydropterin have been defined 7,8-dihydropterin (5), quinonoid dihydropterin (6), 5,6-dihydropterin (7) and 5,8-dihydropterin (8). Of these, only 7,8-dihydropterin derivatives can be stored for long periods under non-aerobic conditions. Indeed, several 7,8-... 

Modeling of the pterin-dithiolate center in Mo enzymes has provided the spur for much work of oxodithiolene complexes in which the metal is formally in oxidation state VI. This included the synthesis of [Mo02(mnt)2] , [M0O2 S2C2(CO2Me)2 2]2-, [Mo02(bdt)2]2-, [Mo02(S2... 

Molybdopterin itself is also extremely unstable when released from a protein and has never been structurally characterized in its native state (32, 33). Mass spectral and NMR studies of the difcarboxamido-methyl) derivative of the oxidized form of molybdopterin have provided convincing evidence that this derivative is a 6-substituted pterin that possesses structure 3 (34). A 6-substituted pterin moiety now appears to be a common feature of all of the molybdenum enzymes of Table I. There is still some question about the oxidation state of the pterin ring... 

In summary, a 6-substituted pterin was first identified as a structural component of the molybdenum cofactor from sulfite oxidase, xanthine oxidase and nitrate reductase in 1980 (24). Subsequent studies provided good evidence that these enzymes possessed the same unstable molyb-dopterin (1), and it seemed likely that 1 was a constituent of all of the enzymes of Table I. It now appears that there is a family of closely related 6-substituted pterins that may differ in the oxidation state of the pterin ring, the stereochemistry of the dihydropterin ring, the tautomeric form of the side chain, and the presence and nature of a dinucleotide in the side chain. In some ways the variations that are being discovered for the pterin units of molybdenum enzymes are beginning to parallel the known complexity of naturally occurring porphyrins, which may have several possible side chains, various isomers of such side chains, and a partially reduced porphyrin skeleton (46). 

The metabolism of folic acid involves reduction of the pterin ting to different forms of tetrahydrofolylglutamate. The reduction is catalyzed by dihydtofolate reductase and NADPH functions as a hydrogen donor. The metabolic roles of the folate coenzymes are to serve as acceptors or donors of one-carbon units in a variety of reactions. These one-carbon units exist in different oxidation states and include methanol, formaldehyde, and formate. The resulting tetrahydrofolylglutamate is an enzyme cofactor in amino acid metabolism and in the biosynthesis of purine and pyrimidines (10,96). The one-carbon unit is attached at either the N-5 or N-10 position. The activated one-carbon unit of 5,10-methylene-H folate (5) is a substrate of T-synthase, an important enzyme of growing cells. 5-10-Methylene-H folate (5) is reduced to 5-methyl-H,j folate (4) and is used in methionine biosynthesis. Alternatively, it can be oxidized to 10-formyl-H folate (7) for use in the purine biosynthetic pathway. 

The absorption band at 384 nm is composed of contributions of the radical species and the second chromophore, whereas the fluorescence spectra with excitation maxima at 398 nm and emission maxima at 470-480 nm are attributed to the pterin alone (146, 155). The 7,8-dihydropterin cofactor, Xmax = 360 nm when free in solution and 390 nm when protein bound, is labile at neutral pH, readily decomposing upon denaturation to form products without significant visible absorption maxima. The photoreduction described above also reduces the second cofactor but in an irreversible manner with complete loss of its fluorescence and visible absorption characteristics (157). Reduction of the blue semiquinone FAD cofactor to the fully reduced form has no effect on the absorption spectrum of the pterin, suggesting that the absorption spectrum of the second cofactor must be independent of the oxidation state of the flavin and that the two cofactors are electronically isolated from each other (157). However, reduction of the flavin radical results in an increase in the fluorescence of the second cofactor, possibly indicating that the flavin radical acts as a potent quencher of fluorescence of the 7,8-dihydropterin. 

Enzymes of the sulfite oxidase family coordinate a single equivalent of the pterin cofactor with an MPT-Mo 02 core in its oxidized state (54, Figure 16), and usually an additional cysteine ligand, which is provided by the polypeptide. Members of this family catalyze the transfer of an oxygen atom either to or from the substrate. Among the members of this family are sulfite oxidase, sulfite dehydrogenase, assimilatory nitrate reductases, and the YedY protein, the catalytic subunit of a sulfite oxidase homologue in E. coli So far, all members of this family contain the MPT-form of Moco without an additional dinucleotide. 

The known redox roles of tetrahydropterins in biochemistry led Rajagopalan to investigate the redox behavior of the pterin unit of Moco. They titrated Moco within molybdoproteins (XO and SO) with two different oxidants, ferrocyanide and the redox dye dichlorophenol indophenol (DCIP), and obtained unexpected results two electron equivalents of either oxidant produced the spectral signature of a fully oxidized pterin (Scheme 2.1), a result only consistent with the pterin in Moco starting at the dihydro oxidation state rather than the tetrahydro state as initially proposed. The interpretation at the time was that the pterin in Moco, instead of the initially proposed tetrahydropterin structure, was a dihydropterin in an unusual tautomeric form. 

The earliest period of work on pterin models for Moco followed the discovery of the pterin unit within Moco, and occurred prior to the confirmation of the dithiolene chelate. These early studies explored the coordination chemistry between molybdenum and pterins or other structurally related molecules such as pteridines (Figure 2.1, top). The resulting themes of this body of work include the favorable coordination by molybdenum in several oxidation states to the 04, N5 chelate site in pterin (see Figure 2.1 for numbering), a variety of reactivities exhibited by Mo -tetrahydropterin systems and the highly delocalized electronic structures in molybdenum-pterin complexes that defy formal oxidation state assignments to Mo and pterin. 

Subsequent Mo-pterin model investigations addressed a seeond provocative aspeet of Rajagopalan s proposed Moeo strueture. The 1982 proposal for Moco paired an oxidized Mo center with a redueed tetrahydropterin - a juxtaposition of the highest molybdenum oxidation state with the most reduced form of pterin - that seemed incompatible and implied possible redox reactions between the metal and the organie eofactor. This seetion deseribes studies direeted at exploring whether molybdenum and pterin redox reactions might occur. These studies were conducted from 1989 to 1999 and inelude reactivity studies of oxidized molybdenum(vi) with redueed pterins, and studies of redueed molybdenum(iv) with oxidized pterins and pteridines. 

The fully oxidized state, the semi-redueed or dihydro state and the fully reduced or tetrahydro state (Seheme 2.2a-e) are the three main redox states of pterins interconverted by 2e , 2H reaetions. The complexity of pterin redox chemistry results from many tautomeric forms of the semi-redueed state (Scheme 2.2d-h), whieh, unless highly substituted, will eventually rearrange to the most thermodynamieally stable form, ca. the 7,8-dihydropterin (Scheme 2.2b)... 

Scheme 2.2 The three oxidation states of pterin (a-c, top row) and the multiple dihydro pterin tautomers (d-h) that are less thermodynamically stable.

Pterin-Molybdenum Redox Chemistry. The early studies focused on Mo(vi) and tetrahydropterin reagents to closely mimic the Mo and pterin portions of Moco. These studies showed that a variety of dioxo-Mo(vi) complexes reacted with tetrahydropterins to produce intensely colored mono-0x0 Mo complexes, but the reports offered different interpretations of what reaction had occurred and what resulting oxidation states of molybdenum and pterin had been produced, even when X-ray structures of the product Mo-pterin complexes were available. A clear picture of the electronic structure was eventually developed from redox titrations, reactivity studies, theoretical calculations and X-ray photoelectron spectroscopy (XPS) studies. 

The second method of establishing oxidation states employed redox titration using the redox dye dichlorophenolindophenol (DCIP) based on the knowledge that tetrahydropterins reduce DCIP instantaneously while qui-nonoid dihydropterins react slowly and 7,8-dihydropterins do not reduce DCIP at all. As previously mentioned in this chapter, DCIP oxidation of Moeo within several molybdoenzymes was determined to be a two-electron process, suggesting a dihydropterin reduction state that was speculated to be the quinonoid tautomer. The results of stoichiometric additions of DCIP to the molybdenum complexes of reduced pterins showed that no oxidation of... 

Formal Oxidation States in Molybdenum-Pterin Complexes. 

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