Stabilization of the Benzoquinoid Anionic Form of 6 and 8-Substituted Hydroxy-

The use of modified flavin chromophores at the active sites of many flavoenzymes is a powerful tool to probe the protein microenvironment of the bound flavins (2). The presence of a positively charged amino acid or a dipole of an α-helix at the active site of many flavoprotein oxidases was proposed long before the crystal structures of enzymes became available (6, 14, 18- 20). This proposal was mainly based on the use of modified flavin chromophores that can exist in different tautomeric and/or mesomeric forms, such as 6- and 8- hydroxy and mercaptoflavins. As an example, 8-Mercaptoflavin has significant spectral differences between the neutral and the anionic species, as well as between the corresponding benzenoid and paraquinoid forms (Figure 1.8) (2). The spectral properties of these chromophores were confirmed by binding to flavoproteins with known crystal structure, such as flavodoxin, glutathione reductase, p- hydroxybenzoate hydroxylase, glucose oxidase, and riboflavin-binding protein (62-65). For both glutathione reductase and p-hydroxybenzoate hydroxylase, in which a positive charge from the α-helix dipole is oriented toward the N(1)-C(2)=O locus of the bound flavin (63-65), the expected spectrum of the anionic paraquinoid species of 8-mercaptoflavin (species D, Figure 1.8) was obtained. Similarly, for flavodoxin, in which the flavin C(8) locus is solvent accessible (66), the expected spectrum of the anionic paraquinoid species of 8-mercaptoflavin (species C, Figure 1.8) was also obtained. The most significant effects of binding of 8-mercaptoflavin to the proteins were observed with both glucose oxidase (Figure 1.9) and riboflavin-binding protein. While in glucose oxidase the anionic paraquinoid resonance (species D, Figure 1.8) was stabilized, a typical spectrum corresponding to the neutral benzenoid form (species A, Figure 1.8) was obtained upon the binding of 8-mercaptoflavin to riboflavin-binding protein, consistent

with the preference of this protein to bind neutral flavin (67). The anionic paraquinoid form of 8- mercaptoflavin was also observed upon the binding of 8-mercaptoflavin to other flavoprotein oxidases, such as D-amino acid oxidase, lactate oxidase, and old yellow enzyme (18).

N N N N HS O O H H3C R N N N H N S O O H H₃C R N N N N -_S O O H H₃C R N N N N S O O H H₃C R + H+ pK ~3.8 A- λ_max ~470 nm B- λ_max ~560 nm C- λ_max ~520 nm D- λ_max ~560-600 nm

Figure 1.8. Tautomeric and mesomeric forms of 8-mercaptoflavin.

A, neutral benzenoid species; B, neutral paraquinoid species; C, anionic benzenoid species (8- thiolate); D, anionic paraquinoid species. Modified from ref. (2).

Figure 1.9. Spectral changes upon binding of 8-mercapto-FAD to the apoprotein of glucose oxidase.

Solid curve, 8-mercapto-FAD; broken curve, bound to apo-glucose oxidase. Taken without permission from ref. (18).

Overall, by the use of 8-mercaptoflavin as a probe of the flavin microenvironment in many flavoproteins, three conclusions have been drawn. 1) The 8-thiolate (anionic) form of 8- mercaptoflavin (species C, Figure 1.8) is stabilized by flavoproteins that catalyze one-electron transfer reactions, such as flavodoxin and NADPH-cytochrome P-450 reductase (2, 6, 18). These proteins also stabilize the neutral (blue) semiquinone species upon reduction. 2) The anionic paraquinoid form of 8-mercaptoflavin (species D, Figure 1.8) is stabilized by flavoproteins of the oxidase/dehydrogenase class, such as glucose oxidase, lactate oxidase, D-amino acid oxidase, and old yellow enzyme (2, 6, 18). These proteins also stabilize the red anionic form of semiquinone and the flavin-N(5)-sulfite adduct as well. 3) Other flavoproteins do not conform to the classification above, showing properties of both classes, such as flavoprotein transhydrogenases and hydroxylases (Table 1.1) (18).

The effect of binding of 8-hydroxyflavins on flavoproteins could be explained by following the same principles and structures used with 8-mercaptoflavin. However, the spectral changes attributed to the ionized paraquinoid species of 6-hydroxyflavin are consistent with the charge being localized in the pyrimidine ring (8). This could be attributed to the difference between oxygen and sulfur in electronegativity and in undergoing π-interactions (2).

The effects of binding of 6-hydroxy- and 6-mercaptoflavin to flavoproteins could be also described by the same structures and reasoning used for 8-substituted analogs. In solution, while the neutral species is protonated at C(6) locus, the anionic form exhibits a longer (red shifted) wavelength absorbance spectrum, consistent with N(1)-blocked 6-hydroxyflavins (Figure 1.10) (9, 67, 68). Therefore, it has been suggested that the anionic paraquinoid species of 6- hydroxyflavin can provide significant information about the charge distribution around the bound flavin (2).

The microenvironment of the bound flavin could be also probed by monitoring the shifts in pKa values of these oxidized flavin analogs upon binding to apoproteins (the pKa values of 8-

SH, 8-OH, 6-SH, and 6-OH flavin analogs are 3.8, 4.8, 5.9, and 7.1, respectively) (2).

N N N N O O H R XH N N N N O O H R X - H+ X = O or S

Figure 1.10. Structures of the neutral and the anionic form of 6-hydroxy- and 6-mercaptoflavins. Modified from ref. (2).

In conclusion, from the use of 6- and 8-sustituted flavin analogs, along with the stabilization of the anionic flavin semiquinone radical or hydroquinone, and the flavin-N(5)- sulfite adduct as different probes for the flavin microenvironment of many flavoprotein before their X-ray crystal structures become available, it has been proposed that a flavoprotein with a positive charge (amino acid residue or α-helix dipole) in the vicinity of the flavin N(1) locus should: 1) stabilize the anionic semiquinone as well as the hydroquinone species of flavin; 2) stabilize a reversible flavin-N(5)-sulfite adduct; 3) stabilize the benzoquinoid anion form of 8- mercaptoflavin; 4) lower the pKa of the 6- and 8-substituted flavin analogs. Most interestingly, it

was also proposed that protein-flavin interaction would enhance the uptake of the redox equivalents through position N(5) during catalysis, thereby a contribution on the redox potential of the enzyme-bound flavin should be also observed as well (18, 20, 26, 27, 69).