Tetrahydrobiopterin as a Trigger for Vitiligo: Phototransformation during UV Irradiation

Vitiligo is a type of hypomelanosis. Tetrahydrobiopterin (H4Bip), the coenzyme of the initial stage of melanogenesis, appears to be a trigger for vitiligo. H4Bip is present in vitiligo in 3–5-fold excess and causes oxidative stress by triggering an autocatalytic cycle of excess hydrogen peroxide synthesis. Using quantum-chemical calculations, we have evaluated the possibility of H4Bip reactions occurring in the dark and under ultraviolet (UV) irradiation, including the formation of dihydropterin dimers. In order to simulate the oxidative stress, oxidative modification of human serum albumin (HSA) has been carried out in the presence of excessive H4Bip using the fluorescence method. The fraction of oxidized protein (FOP) has been calculated. It has been established that there is a strong oxidative modification of amino acids chromophores (tryptophan and tyrosine) in the protein (FOP 0.64). Under UV irradiation of the system (HSA + H4Bip), FOP is reduced to 0.39. Apparently, a part of H4Bip transforms into dihydropterin dimers and does not participate in the oxidative modification of the protein. The data on oxidative modification of HSA are consistent with dynamic light scattering: H4Bip promotes HSA aggregation with the formation of particles with a hydrodynamic radius Rh ≥ 2000 nm, which can become immunogenic.


Introduction
Vitiligo is a chronic dermatological disease characterized by the formation of depigmented patches on the skin due to impaired melanin pigment biosynthesis [1][2][3].The incidence of vitiligo varies by country, from 0.1% to 4.0% [4][5][6][7].An increase in the incidence of this disease is observed, which determines the relevance of this study.
Figure 2. Scheme of tetrahydrobiopterin autoxidation and autocatalytic cycle closure in vitiligo (GTP-guanosine triphosphate; qH2Bip-quinonoid 6,7-dihydrobiopterin; H2Bip-7,8-dihydrobiopterin; H2Ptr-7,8-dihydropterin; Bip-biopterin; H2Xap-dihydroxanthopterin).The long-wavelength maxima of the absorption spectra are indicated in brackets [28].The formation of the H 4 Bip oxidized derivatives is accompanied by the formation of hydrogen peroxide (H 2 O 2 ) (Figure 2).Rancy et al. (2020) [26] reviewed 5147 publications in which H 2 O 2 was used to trigger oxidative stress.It was observed that in half of the publications, the H 2 O 2 concentration in the range of 100-500 µmol was used to trigger cellular oxidative stress.In vitiligo, H 2 O 2 was identified in the skin of patients in millimolar concentrations [15,18,19,23,27], which means that there is strong oxidative stress.The formation of the H4Bip oxidized derivatives is accompanied by the formation of hydrogen peroxide (H2O2) (Figure 2).Rancy et al. (2020) [26] reviewed 5147 publications in which H2O2 was used to trigger oxidative stress.It was observed that in half of the publications, the Н2О2 concentration in the range of 100-500 µmol was used to trigger cellular oxidative stress.In vitiligo, Н2О2 was identified in the skin of patients in millimolar concentrations [15,18,19,23,27], which means that there is strong oxidative stress.
We have shown that ultraviolet (UV) irradiation enhances the oxidation of H 4 Bip, both due to the excitation of H 4 Bip itself (λ max 298 nm) and the excitation of biopterin (Bip, λ max 346 nm), the H 4 Bip oxidation product.Irradiation in the absorption region of Bip triggers the process of photosensitized oxidation of H 4 Bip.Such photooxidation of H 4 Bip can lead to the accumulation of an additional amount of H 2 O 2 and thus prolong vitiligo [29][30][31].
UVB (280-320 nm) phototherapy using UV at 308 and 311 nm is the most successful phototherapy for vitiligo [18,32], but the mechanism of the therapeutic effect has long remained unclear.We have previously proposed a hypothesis and presented arguments in its favor, according to which the main target of UVB radiation is H 4 Bip [33].The formation of dimers of the azocyclobutane type during UVB photooxidation of H 4 Bip has been shown for the first time.The photoformation of dihydropterin dimers ((H 2 Ptr) 2 ) from H 4 Bip has been proven, and it has been shown that the action spectrum of UV radiation lies in the region of 300-325 nm [28].
The study of the H 4 Bip autoxidation process and the literature data on its excessive synthesis in vitiligo allowed us to conclude that the pathology of vitiligo is based on the formation of an autocatalytic cycle of excessive H 2 O 2 synthesis (Figure 2).Under conditions of oxidative stress, H 2 O 2 can trigger an autocatalytic cycle through cytokines, in particular γ-interferon.γ-interferon activates the interferon-inducible enzyme, GTP-cyclohydrolase I (EC 3.5.4.16), which synthesizes an excess of H 4 Bip.UVB phototherapy of vitiligo will break the autocatalytic cycle of excess H 2 O 2 synthesis due to the removal of excess H 4 Bip in the form of (H 2 Ptr) 2 dimers.Therefore, such UVB therapy will help to restore the process of melanogenesis.
Since autoxidation and photooxidation of H 4 Bip are radical processes, in this paper, using the methods of quantum chemistry, a theoretical study of the possibility of the occurrence of reactions involved in the processes of impaired melanogenesis and UV therapy of vitiligo was carried out.In addition to studying the radical processes of autoxidation and photooxidation of H 4 Bip in buffer solutions, reactions were carried out in the presence of human serum albumin (HSA) to analyze the effect of radicals on proteins, including melanogenesis enzymes.HSA was used as a model for the H 4 Bip protein environment under oxidative stress conditions in cells.Further development in this direction is necessary to improve the methods for diagnosing and treating vitiligo.•− ) production [34]:

Results
Autoxidation can also be initiated with the production of trihydrobiopterin radical (H 3 Bip • ) and hydroperoxyl radical (HOO • ): O 2 •− and HOO • , which are produced in Reactions (1) and ( 2), do take part in the oxidation of H 4 Bip.As was previously established, the oxidation of H 4 Bip in the presence of O 2 in aqueous solutions has a radical chain character [34].The main products of H 4 Bip autoxidation are well-known.However, the nature and the role of short-lived intermediate free radical species is debatable.The same is true for the photooxidation of H 4 Bip.
As a result, the Ptr •− radical anion and the H 4 Bip •+ radical cation are produced.This reaction relates to type-I sensitization mechanism: Pterin triplets can oxidize not only H 4 Bip, but also other electron donors.Electron transfer from an enzyme to a pterin triplet can inactivate the enzyme, in particular tyrosinase [36], which becomes a significant problem in vitiligo disease.
We obtained the vertical ionization potential (VIP) of H 4 Bip to test the legitimacy of our theoretical approach.According to the MP2/6-31G(d,p)+ calculation, VIP is equal to 4.62 eV.The value of 4.82 eV was estimated using the dielectric constant of solvent, epsilon, which was equal to 80, in the study by Gogonea et al. [37].According to the calculations carried out using the M06-2X/6-31G(d,p)+ method, VIP was equal to 4.73 eV [38].Therefore, our MP2/6-31G(d,p)+ calculations are in agreement with previous studies.VIP is an important characteristic which determines various pterin properties, including singlet oxygen generation efficacy [30].In addition, we determined the vertical electron affinity (VEA) of 3 O 2 .According to our calculations, it is equal to −3.43 eV, whereas the experimentally determined value is equal to −3.5 eV [39].In case of the M06-2X calculations, the VEA of molecular oxygen was equal to −3.41 eV [38].For this reason, our MP2/6-31G(d,p)+ method using the COSMO solvent model is an adequate and legitimate approach.
The experimental study of free radical species and their reactions requires application of techniques such as electron paramagnetic resonance, which is time-and labor-consuming.However, quantum chemistry methods allow for evaluating the feasibility of such reactions theoretically.We started with the feasibility estimation for the reaction between H 4 Bip and molecular oxygen (Reaction (1)).The Gibbs free energy of reactions (∆ f Gº) is equal to 26.5 kcal mol −1 , according to our calculations.This means that Reaction (1) does not occur spontaneously.
Reaction (2) occurs between the same reagents as Reaction (1) and produces other radicals: H 3 Bip • and HOO • .Production of H 3 Bip • and HOO • should not occur as well: ∆ f Gº is equal to 30.8 kcal mol −1 .Nevertheless, H 3 Bip • and HOO • are not the final products.Next, the quinonoid 6,7-dihydrobiopterin (qH 2 Bip) and H 2 O 2 formation occurs: The Gibbs free energy of Reaction (4) is −4.3 kcal mol −1 , which means that the reaction between H 4 Bip and molecular oxygen could take place spontaneously.Since 7,8-dihydrobiopterin (H 2 Bip) is 15.3 kcal mol −1 more stable than qH 2 Bip, the final pair of products of the reaction between H 4 Bip and molecular oxygen should be H 2 Bip and hydrogen peroxide: Reaction (5) possesses a Gibbs free energy equal to −19.5 kcal mol −1 .Figure 3 shows the outline of the reaction process between H 4 Bip and molecular oxygen.The energetic characteristics of the intermediate products are insignificant, since only the Gibbs free energy of initial reagents and final products is important (−19.5 kcal mol −1 ).For this reason, the autoxidation of H 4 Bip does occur spontaneously.As a whole, there exist a general force for H 4 Bip autoxidation, despite several uphill elementary reactions.Uphill reactions do take place, but possess slow kinetics: in particular, the experimental rate constant of Reaction (1) is equal to 0.6 M −1 s −1 [34].There is an alternative to H2Bip formation during Reaction (5): 7,8-dihydropterin (H2Ptr) and 2-hydroxypropanal can also be formed.However, these products are 5.1 kcal mol −1 less stable than H2Bip, which is confirmed with the experiments conducted in phosphate buffer pH 7.3 [29].
The H4Bip free radical species also take part in autoxidation.Thus, H3Bip • and molecular oxygen participate as educts in Reaction (8): This reaction is a rate limiting step in H4Bip prolonged oxidation with a reaction rate constant equal to 3.2 × 10 3 M −1 s −1 [34].The reaction is possible since its Gibbs free energy is negative (−1.5 kcal mol −1 ).The production of a short-living complex between H3Bip • and O2 is also feasible [34]: However, Reaction (9) possesses a positive ∆fGº value (13.5 kcal mol −1 ).It is still feasible, since the overall reaction (Reaction ( 8)) has a negative ∆fGº.
The trihydrobiopterin radical, along with O2 •− , can participate in the production of the H3BipOO − intermediate complex due to Reaction (10): which possesses a Gibbs free energy value equal to −15.7 kcal mol −1 .Then, the short-living intermediate complex can be protonated: Reaction (11) possesses a significantly negative Gibbs free energy (−42.9kcal mol −1 ).Reactions ( 1), ( 2), ( 6) and ( 7) relate to the initiation of the free radical chain process, whereas Reactions ( 8)-( 10) relate to the chain elongation.Reactions of free radical chain There is an alternative to H 2 Bip formation during Reaction (5): 7,8-dihydropterin (H 2 Ptr) and 2-hydroxypropanal can also be formed.However, these products are 5.1 kcal mol −1 less stable than H 2 Bip, which is confirmed with the experiments conducted in phosphate buffer pH 7.3 [29].
During H 4 Bip autoxidation, it can be oxidized by reactive oxygen species (ROS).See, for example, Reactions ( 6) and ( 7): The rate constant of Reaction ( 6) is known to be 3.9 × 10 5 M −1 s −1 [34]; therefore, it is extremely fast.The Gibbs free energy of Reaction (6) is 7.3 kcal mol −1 ; therefore, it is unfavorable.On the contrary, a synonymic reaction, Reaction (7), is feasible: The H 4 Bip free radical species also take part in autoxidation.Thus, H 3 Bip • and molecular oxygen participate as educts in Reaction (8): This reaction is a rate limiting step in H 4 Bip prolonged oxidation with a reaction rate constant equal to 3.2 × 10 3 M −1 s −1 [34].The reaction is possible since its Gibbs free energy is negative (−1.5 kcal mol −1 ).The production of a short-living complex between H 3 Bip • and O 2 is also feasible [34]: However, Reaction (9) possesses a positive ∆ f Gº value (13.5 kcal mol −1 ).It is still feasible, since the overall reaction (Reaction ( 8)) has a negative ∆ f Gº.
The trihydrobiopterin radical, along with O 2 •− , can participate in the production of the H 3 BipOO − intermediate complex due to Reaction (10): which possesses a Gibbs free energy value equal to −15.7 kcal mol −1 .Then, the short-living intermediate complex can be protonated: Reaction (11) possesses a significantly negative Gibbs free energy (−42.9kcal mol −1 ).Reactions ( 1), ( 2), ( 6) and ( 7) relate to the initiation of the free radical chain process, whereas Reactions ( 8)-( 10) relate to the chain elongation.Reactions of free radical chain termination are also a part of the H 4 Bip autoxidation process.In particular, the reaction between two H 3 Bip • molecules is feasible both experimentally [34] and theoretically: The rate constant of Reaction ( 12) is 9.
The free radical chain process can be terminated when its reactive oxygen species interplay with other ROS.Here are a few reactions: The Gibbs free energy of Reaction ( 13) is −48.9 kcal mol −1 , and therefore, it is feasible.Reaction (14) possesses ∆ f Gº equal to −23.6 kcal mol −1 and is also favorable.
Thus, we have considered 14 reactions taking place during H 4 Bip oxidation in the presence of O 2 .All reactions are possible from the viewpoint of the MP2/6-31G(d,p)+ calculation, except for Reaction (6) (formation of H 3 Bip • and HOO − when H 4 Bip and O 2 •− are used as educts).

Type-I H 4 Bip Photooxidation
Along with autoxidation under UV irradiation, tetrahydrobiopterin can also be subjected to photooxidation [29,33].Oxidized pterins are used as photosensitizers in this process.Oxidized pterins are the final products of H 4 Bip oxidation in aqueous solutions.Pterin (Ptr) forms a singlet excited state ( 1 Ptr*) under UV radiation; then, production of 3 Ptr* is possible through the intersystem crossing: Oxidation of H 4 Bip by the pterin triplets occurs predominantly (87%) through the type-I photosensitization mechanism [29].It starts with the reaction between H 4 Bip and 3 Ptr* (Reaction (3)).
The Gibbs free energy of Reaction (3) is negative (−21.4 kcal mol −1 ) and favorable.Thus, the type-I sensitization mechanism begins with the production of H 4 Bip •+ and Ptr •− radicals.In air-equilibrated solutions, Reaction (16), which is the electron transfer from Ptr •− to O 2 , can also occur: This reaction is possible because the Gibbs free energy of such a reaction is equal to −30.4 kcal mol −1 .O 2 •− can then oxidize H 4 Bip.Direct electron transfer from 3 Ptr* to O 2 and the O 2 •− formation, however, are unfavorable since ∆ f Gº of this reaction is positive and equals to 18.6 kcal mol −1 .
The type-I sensitization mechanism is a part of the free radical chain process of both H 4 Bip autoxidation ("dark" reactions) and photooxidation (Table 1).(13) chain termination −48.9 Thus, we have evaluated the possibility of more than 15 reactions that relate to H 4 Bip oxidation.The conclusions about the feasibility of certain "dark" and light reactions are in line with our previous studies [31,38].Next, we turn to the reactions of the dihydropterin dimer formation.

Dihydropterin Dimer Formation
In our previous study [33], we found that the azacyclobutane cis isomer (Figure 4) is the most favorable product of dihydropterin photodimerization.However, it is not clear which molecules participate in the dimerization: a quinonoid 6,7-H 2 Ptr (qH 2 Ptr) and benzoid form of 7,8-H 2 Ptr, ground state molecules or excited state triplets?To answer these questions, we have performed quantum-chemical calculations for the respective H 2 Ptr molecules.Thus, we have evaluated the possibility of more than 15 reactions that relate to H4Bip oxidation.The conclusions about the feasibility of certain "dark" and light reactions are in line with our previous studies [31,38].Next, we turn to the reactions of the dihydropterin dimer formation.

Dihydropterin Dimer Formation
In our previous study [33], we found that the azacyclobutane cis isomer (Figure 4) is the most favorable product of dihydropterin photodimerization.However, it is not clear which molecules participate in the dimerization: a quinonoid 6,7-H2Ptr (qH2Ptr) and benzoid form of 7,8-H2Ptr, ground state molecules or excited state triplets?To answer these questions, we have performed quantum-chemical calculations for the respective H2Ptr molecules.The reaction does not occur since its ∆fGº is positive and equals to 5.9 kcal mol −1 .However, when one of the H2Ptr molecules is the qH2Ptr isomer, Reaction ( 18) is feasible because ∆fGº equals to −6.6 kcal mol −1 : H2Ptr + qH2Ptr → (H2Ptr)2 (18) qH2Ptr is 12.5 kcal mol −1 less stable than H2Ptr.∆fGº of the reaction equals to −19.2 kcal mol −1 when two qH2Ptr molecules are used as educts.Though Reaction (18) is feasible according to the calculations, it is not confirmed experimentally [33]: the dimers are not formed during the autoxidation of H4Bip.The reason is probably a low lifetime of the quinonoid and its conversion to benzoic dihydropterin.First, we have evaluated the feasibility of the dimerization of two ground state molecules: The reaction does not occur since its ∆ f Gº is positive and equals to 5.9 kcal mol −1 .However, when one of the H 2 Ptr molecules is the qH 2 Ptr isomer, Reaction ( 18) is feasible because ∆ f Gº equals to −6.6 kcal mol −1 : H 2 Ptr + qH 2 Ptr → (H 2 Ptr) 2 (18) qH 2 Ptr is 12.5 kcal mol −1 less stable than H 2 Ptr.∆ f Gº of the reaction equals to −19.2 kcal mol −1 when two qH 2 Ptr molecules are used as educts.Though Reaction (18) is feasible according to the calculations, it is not confirmed experimentally [33]: the dimers are not formed during the autoxidation of H 4 Bip.The reason is probably a low lifetime of the quinonoid and its conversion to benzoic dihydropterin.∆ f Gº is −58.4 kcal mol −1 when one of the H 2 Ptr molecules is in the triplet state: Therefore, Reaction (19) is favorable.If one of the molecules is a quinonoid, ∆ f Gº is even lower (−65.9kcal mol −1 ): Reaction ( 21) is even more feasible than Reaction (20): the Gibbs free energy equals to −71.0 kcal mol −1 .Thus, Reactions ( 19)-( 21) are all favorable.However, in real experimental conditions, the situation is different: when H 2 Bip is used as an educt of photodimerization, the quantum yield is around 5% [40].However, when the initial reagent is H 4 Bip, the quantum yield of the dimer formation is ca.96% [33].In this regard, Reaction (21) looks like the most favorable among Reactions ( 19)-( 21).The energy profiles of all the described reactions are summarized in Figure 5. Reaction ( 21) is even more feasible than Reaction (20): the Gibbs free energy equals to −71.0 kcal mol −1 .Thus, Reactions ( 19)-( 21) are all favorable.However, in real experimental conditions, the situation is different: when H2Bip is used as an educt of photodimerization, the quantum yield is around 5% [40].However, when the initial reagent is H4Bip, the quantum yield of the dimer formation is ca.96% [33].In this regard, Reaction (21) looks like the most favorable among Reactions ( 19)-( 21).The energy profiles of all the described reactions are summarized in Figure 5.The quinonoid dihydropterin formed during H4Bip autoxidation is a highly active compound of quinoid nature and, apparently, forms an intermolecular complex with dihydropterin, the so-called quinhydrone.In quinhydrones, a weak donor-acceptor bond is formed between the quinoid and benzenoid structures.Being bound into an intermolecular complex (H2Ptr-qH2Ptr), the constituent components are not able to react with each other in the ground state, but are able to react in an excited state when exposed to light.The dimerization reaction is apparently facilitated by the mutual orientation of the quinonoid and benzoic forms of dihydropterins in the complex.The quinonoid dihydropterin formed during H 4 Bip autoxidation is a highly active compound of quinoid nature and, apparently, forms an intermolecular complex with dihydropterin, the so-called quinhydrone.In quinhydrones, a weak donor-acceptor bond is formed between the quinoid and benzenoid structures.Being bound into an intermolecular complex (H 2 Ptr-qH 2 Ptr), the constituent components are not able to react with each other in the ground state, but are able to react in an excited state when exposed to light.The dimerization reaction is apparently facilitated by the mutual orientation of the quinonoid and benzoic forms of dihydropterins in the complex.

H 4 Bip Irradiation in the Presence and Absence of HSA
Figure 6 shows the results of an experiment on irradiating the H 4 Bip solution in the presence of HSA.As can be seen, during irradiation, the absorption increases in the region of 242 nm, which indicates the formation of dihydropterin dimers (with a yield of about 40 ± 1.8%).In the control without irradiation, H 4 Bip is oxidized only to H 2 Bip and dihydropterin, which coincides with the initial stages of H 4 Bip autoxidation, as shown in Figure 2.  It can be seen from the difference absorption spectrum (Figure 6, inset) that upon irradiation, the compound with the absorption maximum at 307 nm decreases, while the absorption increases in the region of 242 nm, and an inflection appears in the region of 276 nm.This may indicate a decrease in the intermediate intermolecular complex (qH2Ptr-H2Ptr) and an increase in the amount of the resulting dimer (H2Ptr)2.An intermediate intermolecular complex can be formed during donor-acceptor interactions of the benzoid form of dihydropterins (H2Ptr or H2Bip) with the quinonoid form of dihydropterins (qH2Ptr or qH2Bip).Using derivative UV spectrophotometry, it was found that the resulting intermolecular complex has maximum absorption at 307 nm, which is found in the spectrum of the fourth derivative of the H4Bip spectrum after keeping the solution in the presence of atmospheric oxygen for several minutes [41].

Oxidative Modifications of HSA in the Presence of H4Bip
Figure 7 shows an experiment on the irradiation of the H4Bip solution in a Tris buffer pH 7.0 with stepwise irradiation at 308 ± 10 nm in the presence of atmospheric oxygen.As can be seen, the dihydropterin dimer is formed with a 44 ± 2.1% yield, which practically coincides with the yield of dimer formation upon the irradiation of H4Bip in the presence of HSA (40 ± 1.8%).This suggests that the presence of a protein has almost no effect on dimer formation.It can be seen from the difference absorption spectrum (Figure 6, inset) that upon irradiation, the compound with the absorption maximum at 307 nm decreases, while the absorption increases in the region of 242 nm, and an inflection appears in the region of 276 nm.This may indicate a decrease in the intermediate intermolecular complex (qH 2 Ptr-H 2 Ptr) and an increase in the amount of the resulting dimer (H 2 Ptr) 2 .An intermediate intermolecular complex can be formed during donor-acceptor interactions of the benzoid form of dihydropterins (H 2 Ptr or H 2 Bip) with the quinonoid form of dihydropterins (qH 2 Ptr or qH 2 Bip).Using derivative UV spectrophotometry, it was found that the resulting intermolecular complex has maximum absorption at 307 nm, which is found in the spectrum of the fourth derivative of the H 4 Bip spectrum after keeping the solution in the presence of atmospheric oxygen for several minutes [41].
Figure 7 shows an experiment on the irradiation of the H 4 Bip solution in a Tris buffer pH 7.0 with stepwise irradiation at 308 ± 10 nm in the presence of atmospheric oxygen.As can be seen, the dihydropterin dimer is formed with a 44 ± 2.1% yield, which practically coincides with the yield of dimer formation upon the irradiation of H 4 Bip in the presence of HSA (40 ± 1.8%).This suggests that the presence of a protein has almost no effect on dimer formation.

Study of HSA Oxidative Modification with Fluorescence
The oxidative modification of HSA was assessed based on the decrease in fluorescence in the region of 350 nm (fluorescence of tryptophan and tyrosine) upon excitation at 280 nm.The light with the spectral range of 308 ± 10 nm, used in the treatment of vitiligo [18,42,43], was considered as the source of oxidative stress.Figure 8a shows the HSA fluorescence spectra before and after irradiation at 308 nm for 16 min.As can be seen from Figure 8a, the fluorescence intensity at 350 nm falls down, which indicates that protein oxidation is taking place.Figure 8b shows the fluorescence spectra (excitation at 280 nm) of HSA solutions in the presence of H4Bip before and after irradiation at 308 ± 10 nm, as well as a control

Study of HSA Oxidative Modification with Fluorescence
The oxidative modification of HSA was assessed based on the decrease in fluorescence in the region of 350 nm (fluorescence of tryptophan and tyrosine) upon excitation at 280 nm.The light with the spectral range of 308 ± 10 nm, used in the treatment of vitiligo [18,42,43], was considered as the source of oxidative stress.Figure 8a shows the HSA fluorescence spectra before and after irradiation at 308 nm for 16 min.As can be seen from Figure 8a, the fluorescence intensity at 350 nm falls down, which indicates that protein oxidation is taking place.

Study of HSA Oxidative Modification with Fluorescence
The oxidative modification of HSA was assessed based on the decrease in fluorescence in the region of 350 nm (fluorescence of tryptophan and tyrosine) upon excitation at 280 nm.The light with the spectral range of 308 ± 10 nm, used in the treatment of vitiligo [18,42,43], was considered as the source of oxidative stress.Figure 8a shows the HSA fluorescence spectra before and after irradiation at 308 nm for 16 min.As can be seen from Figure 8a, the fluorescence intensity at 350 nm falls down, which indicates that protein oxidation is taking place.Figure 8b shows the fluorescence spectra (excitation at 280 nm) of HSA solutions in the presence of H4Bip before and after irradiation at 308 ± 10 nm, as well as a control Figure 8b shows the fluorescence spectra (excitation at 280 nm) of HSA solutions in the presence of H 4 Bip before and after irradiation at 308 ± 10 nm, as well as a control without irradiation for the same time.As can be seen, the fluorescence intensity of the protein solution at 350 nm decreases, while the fluorescence in the region of 450 nm, corresponding to strongly fluorescent oxidized forms of H 4 Bip, increases.
The degree of oxidative modification of HSA, expressed through the fraction of oxidized protein (FOP), is presented in Table 2.As can be seen from Table 2, the greatest changes in the oxidation state of HSA (0.64 ± 0.028) occur when H 4 Bip is added to HSA, i.e., under H 4 Bip autoxidation conditions.These conditions simulate the conditions for the formation of H 2 O 2 in vitiligo.An additional source of radicals to ROS is H 4 Bip, since radicals of a pterin nature are formed during autoxidation.They can serve as an additional source of oxidative stress for the protein.When the system (H 4 Bip + HSA) is irradiated, the degree of protein oxidation is lower (0.39 ± 0.017).The lowest degree of oxidation (0.24 ± 0.01) occurs under the direct action of UV (308 ± 10 nm) on the protein, apparently due to the excitation of protein chromophores (tryptophan and tyrosine).HSA aggregation as a result of oxidative stress in the presence of H 4 Bip without irradiation and under UV irradiation (308 ± 10 nm) was studied using dynamic light scattering (DLS).Figure 9 presents the distributions of hydrodynamic radii (R h ) of protein particles in the studied samples.
It should be noted that, at the studied low protein concentration (0.1 mg/mL), the distributions reflect only the aggregates present in the solution or, in the case of native HSA, oligomers (186 ± 18 nm) (Figure 9a), since small molecules of HSA itself make a low contribution to the light scattering intensity.However, we can observe the formation of larger aggregates with sizes reaching hundreds of nanometers and micrometers (≥2000 nm) upon the addition of H 4 Bip (Figure 9b,c).In the presence of an excess of H 4 Bip (Figure 9b), aggregates with R h 514 ± 119 nm were formed, while under the action of UV in the presence of H 4 Bip (Figure 9c), a part of the protein was preserved in the form of smaller oligomers (R h 221 ± 32 nm).This may be due to the fact that the effect of pterin radicals on HSA is reduced due to the formation of dihydropterin dimers from H 4 Bip.The existence of an additional peak with R h 47 ± 5 nm (Figure 9b) can also be due to increased aggregation of native HSA in the presence of H 4 Bip.It is remarkable that this peak is absent after UV irradiation in the presence of H 4 Bip.

Discussion
We believe that the pathology of vitiligo is based on the formation of an autocatalytic cycle of excessive synthesis of H 2 O 2 , leading to oxidative stress and triggering of melanocyte apoptosis.Where does the excess H 2 O 2 come from?It can occur with an excessive H 4 Bip, which in vitiligo is 3-5 times higher than normal and inevitably undergoes autoxidation in the presence of molecular oxygen.This results in the formation of radical intermediate products of a pterin nature, oxidized forms of pterins and H 2 O 2 (see Figure 2).H 2 O 2 triggers an autocatalytic cycle for the synthesis of excessive hydrogen peroxide through cytokines, in particular through γ-interferon.This activates the interferoninducible enzyme guanosine triphosphate cyclohydrolase, which synthesizes an excess of H 4 Bip.Thus, strong oxidative stress (10 −3 M H 2 O 2 ) occurs in melanocytes.
Using quantum-chemical calculations, we have evaluated the feasibility of reactions participating in H 4 Bip autoxidation and photooxidation.As a whole, H 4 Bip photodegradation occurs according to both type-I and type-II mechanisms.The conclusions about the feasibility of certain "dark" and light reactions are in line with our previous studies [31,38].Among the reactions of (H 2 Ptr) 2 dimer formation, the reaction between qH 2 Ptr and 3 H 2 Ptr* triplet seems to be the most feasible.The energy profiles of all the described reactions are summarized in Figure 5.
We have simulated the conditions of oxidative stress with an excess of H 4 Bip using HSA as a model of the H 4 Bip protein environment.Currently, the oxidative modification of proteins, and in particular the free radical oxidation of HSA, is being widely studied.HSA is considered as a target for free radicals and as a marker of oxidative stress [44][45][46].Pterin radicals generated during H 4 Bip photooxidation can function as photosensitizers of protein oxidation [47,48].The radical trap in HSA is the thiol S-H group of Cys-34 [49].The second amino acid sensitive to the action of free radicals is methionine.HSA contains six methionine residues.The third target for free radicals in HSA is 18 tyrosines and 1 tryptophan, which fluoresce under UV irradiation [46,50,51].Therefore, it is possible to study the oxidative degradation of amino acids in a protein using a highly sensitive UV fluorescence method.
It has been shown that the fluorescence of HSA in the presence of H 4 Bip and oxygen strongly decreases: FOP is 0.64 ± 0.028, and hence, more than half of the chromophores are subjected to destruction.This seems to be due to the fact that the protein is a target for free radicals of both a pterin nature [52] and ROS, which are generated during H 4 Bip autoxidation.
When the (H 4 Bip+ HSA) system is exposed to UV (308 ± 10 nm), dihydropterin dimers are formed in parallel with the oxidative degradation of the protein by pterin radicals, ROS, and H 2 O 2 .As a result of the formation of dimers, H 4 Bip leaves the system and a smaller amount of it is involved in the autoxidation, and FOP decreases to 0.39.This simulates the redox state during UVB therapy of vitiligo when H 4 Bip leaves the reaction zone, turning into dimers.In the case where only protein is exposed to UV irradiation in the presence of oxygen, we have the lowest FOP (0.24 ± 0.01).Apparently, this is due to the fact that the irradiation source (308 ± 10 nm) had a spectral slit width of 20 nm and had 298 nm radiation, which primarily excited tryptophan residues in the protein and they were oxidized by oxygen.The first electronic absorption band of tryptophan S 0 → S 1 is in the region of 260-310 nm.Thus, using the UV fluorescence method in the (H 4 Bip + HSA) system, we were able to simulate the relative levels of oxidative stress that occur during the autoxidation and photooxidation of H 4 Bip under conditions of it being present in 3-5-fold excess in vitiligo.At the same time, it was noted that UVB radiation at 308-311 nm has the disadvantage, in that it practically captures the protein excitation region and can lead to oxidative degradation of proteins.In this regard, we have proposed a light-emitting diode with λ max 325 nm as a source of radiation for phototherapy of vitiligo [28].
Oxidative modification of proteins usually leads to a change in the conformation of the molecule due to the formation of dityrosines, and the oxidation of tryptophans and cysteines.These destructions lead to the disappearance of the α-helical structure of the protein and the appearance of aggregates.The DLS method was used to study HSA aggregation in the presence of H 4 Bip.Studies of the distribution of hydrodynamic radii of protein particles showed that in the presence of H 4 Bip in the dark and under UV irradiation, HSA aggregation occurs with a significant increase in particle size, up to hundreds of nanometers and micrometers (≥2000 nm) (Figure 9b,c).In the case of HSA, in the presence of an excess of H 4 Bip, the oxidation products which are a source of radicals (Figure 9b), aggregates with R h 514 ± 10 nm were formed, while under the action of UV in the presence of H 4 Bip, a significant part of the protein was preserved in the form of smaller oligomers (R h 221 ± 10 nm).The smaller aggregates of apparently native HSA (R h 47 ± 5 nm) are also formed in the presence of H 4 Bip without irradiation and do not appear under UV irradiation.This may be due to the fact that the effect of pterin radicals on HSA is reduced due to the transition of H 4 Bip into dihydropterin dimers.The HSA aggregates formed, and the aggregates with R h ≥ 2000 nm can become immunogenic particles upon the interaction with pterin radicals and the highly reactive quinonoid form of dihydropterin.It is known that H 2 O 2 triggers the systems of humoral and cellular immunity, which can respond to emerging immunogenic complexes and lead to the death of melanocytes.

Quantum Chemical Calculations: Conformational Analysis, Geometry Optimization, and Hessian Calculation
Conformational analysis of H 4 Bip and its derivatives has been performed in Spartan v. 20 (http://www.wavefun.com,accessed on 30 August 2023).The series of lowenergy conformers has been generated using molecular mechanics, namely the MMFF force field [53].Selection of the most stable conformer has been carried out based on the Hartree-Fock method and second-order Møller-Plesset perturbation theory (MP2), implemented in the ORCA program v. 3.0 [54].Thus, the MMFF-generated geometries of H 4 Bip and its derivatives have been optimized, and the total energy has been calculated using the MP2/6-31G(d)+ method; thus, the most stable conformers have been selected.Then, further optimization and Hessian calculation have been performed using the MP2/6-31G(d,p)+ method to obtain the Gibbs free energy.The COSMO continuum model [55] has been used to take into account a water solvent with a dielectric constant ε equal to 80.
We have used freshly prepared solutions of 0.1 mg/mL (1.5 × 10 −6 M) HSA in 0.01 M phosphate buffer (pH 7.0) in the absence or presence of 1.1 × 10 −4 M H 4 Bip, as well as solutions of 1.6 × 10 −4 M H 4 Bip in 0.01 M Tris buffer (pH 7.0).The concentration of H 4 Bip has been determined based on the molar extinction coefficient (ε 297 = 10,200 M −1 cm −1 for pH 7.0).Irradiation of 1.5 mL solutions has been carried out in cuvettes with an optical path length of 1 cm, in 2 min steps for 16 min, at 308 ± 10 nm (41.4 W•m −2 ) with constant stirring in air.At each step, the absorption and fluorescence spectra of the studied samples were measured.The FluoroMax 4 spectrofluorimeter (Horiba Scientific, Kyoto, Japan) has been used as an UV light source as described in our previous work [28].Controls without irradiation have been performed in parallel to each experiment.
The absorption spectra of the irradiated samples in the range of 200-500 nm have been recorded using the Shimadzu UV-1601 spectrophotometer (Kyoto, Japan).The concentration of dihydropterin dimers has been determined from the change in the absorption of the band in the 245 nm region and has been calculated taking into account the extinction coefficient (ε 245 = 27,000 M −1 cm −1 for pH 7.0 [28]).
Fluorescence spectra have been recorded upon excitation at 280 nm and extinction at 290-550 nm using the FluoroMax 4 spectrofluorimeter (Horiba Scientific, Kyoto, Japan).In order to assess the degree of protein oxidative modification under these conditions, FOP has been calculated using the formula (I o − I)/I o , where I o is the fluorescence intensity at 350 nm (with excitation at 280 nm) before irradiation, and I is the fluorescence intensity after irradiation or in dark control for the same time.
Dynamic light scattering measurements have been performed using the Photocor Complex correlation spectrophotometer (PhotoCor Instruments, Inc., College Park, MD, USA) equipped with a 632.8 nm He-Ne laser and a temperature controller.Scattered light has been collected at an angle of 90 • .All measurements have been carried out at 25 • C in capped glass vials (40 × 8.2 mm).Protein samples were added to the buffer, preliminarily thermostated in the vial at 25 • C, to the final protein concentration of 0.1 mg/mL.The sample volume was 0.35 mL.The accumulation time for the autocorrelation function was no less than 60 s.The particle size distributions have been calculated using the DynaLS v.2 software.The refraction index of buffer at 25 • C is considered as equal to 1.3324, and the dynamic viscosity is 0.8912 mPa•s.
The experimental results are the arithmetic mean of at least three independent replicates with a 95% confidence interval.The results of the experiment are expressed as mean ± standard deviation.

Conclusions
We have estimated the feasibility of the reactions of H 4 Bip autoxidation and photooxidation in the presence of molecular oxygen from a theoretical viewpoint.We have also considered the reaction of dihydropterin dimer formation upon UV irradiation.The autoxidation occurs according to the experimentally established scheme.Considering the photooxidation of H 4 Bip, we have shown that photooxidation by Ptr is theoretically probable.Table 1 shows the main reactions participating in the photooxidation process: reactions of the radical chain process initiation, prolongation, and termination.Finally, we have evaluated the possibility of H 2 Ptr dimerization reactions.Apparently, the quinonoid form of H 2 Ptr and the triplet state of benzoid form of H 2 Ptr play a significant role in this process.The participation of an intermediate complex of the quinhydrone type, which is formed between the quinonoid and benzoic forms of dihydropterin in the reaction of dimer formation that facilitates this process, is discussed.
Theoretical calculations are consistent with the obtained experimental data, which have shown the formation of dihydropterin dimers under the action of light, regardless of the presence of the protein in the system.Also, the presence of radicals of a pterin nature and ROS during the oxidation of H 4 Bip is confirmed by the fact that oxidative modification of HSA occurs with further aggregation of the oxidized protein, which has been shown using fluorescence methods with FOP calculation and DLS.Under the action of UV irradiation in the presence of H 4 Bip, the degree of oxidative modification of the protein has been lower, which may be due to the phototransformation of H 4 Bip into dihydropterin dimers.It can be assumed that similar processes can occur in a cell during UV phototherapy of vitiligo.The results can be presented in the form of a figure (Figure 10).

Figure 1 .
Figure 1.Melanin biosynthesis and the H 4 Bip regeneration cycle.

Figure 3 .
Figure 3.The Gibbs free energy (∆fGº) profile of the reaction between H4Bip and molecular oxygen.

Figure 3 .
Figure 3.The Gibbs free energy (∆ f Gº) profile of the reaction between H 4 Bip and molecular oxygen.

Figure 5 .
Figure 5. Energy profiles of the H2Ptr dimerization reactions.Photoreactions are considered along with "dark" reactions.

Figure 5 .
Figure 5. Energy profiles of the H 2 Ptr dimerization reactions.Photoreactions are considered along with "dark" reactions.

2. 2 . 1 .
Figure6shows the results of an experiment on irradiating the H4Bip solution in the presence of HSA.As can be seen, during irradiation, the absorption increases in the region of 242 nm, which indicates the formation of dihydropterin dimers (with a yield of about 40 ± 1.8%).In the control without irradiation, H4Bip is oxidized only to H2Bip and dihydropterin, which coincides with the initial stages of H4Bip autoxidation, as shown in Figure2.

Figure 8 .
Figure 8. Changes in the fluorescence spectra of a solution of HSA in 0.01 M phosphate buffer pH 7 (a) in the absence of H4Bip and (b) in the presence of H4Bip and atmospheric oxygen upon excitation at 280 nm: 1-initial solution (0 min); 2-after irradiation at a wavelength of 308 ± 10 nm for 16 min; 3-control without irradiation (16 min).

Figure 8 .
Figure 8. Changes in the fluorescence spectra of a solution of HSA in 0.01 M phosphate buffer pH 7 (a) in the absence of H4Bip and (b) in the presence of H4Bip and atmospheric oxygen upon excitation at 280 nm: 1-initial solution (0 min); 2-after irradiation at a wavelength of 308 ± 10 nm for 16 min; 3-control without irradiation (16 min).

Figure 8 .
Figure 8. Changes in the fluorescence spectra of a solution of HSA in 0.01 M phosphate buffer pH 7 (a) in the absence of H 4 Bip and (b) in the presence of H 4 Bip and atmospheric oxygen upon excitation at 280 nm: 1-initial solution (0 min); 2-after irradiation at a wavelength of 308 ± 10 nm for 16 min; 3-control without irradiation (16 min).

Figure 9 .
Figure 9. Distribution of hydrodynamic radii (Rh) for solutions of 0.1 mg/mL HSA in 0.01 M phosphate buffer, pH 7.0 (a) HSA; (b) HSA in the presence of H4Bip without irradiation; (c) HSA in the presence of H4Bip after irradiation.Irradiation was carried out at 308 ± 10 nm for 16 min.

Figure 9 .
Figure 9. Distribution of hydrodynamic radii (R h ) for solutions of 0.1 mg/mL HSA in 0.01 M phosphate buffer, pH 7.0 (a) HSA; (b) HSA in the presence of H 4 Bip without irradiation; (c) HSA in the presence of H 4 Bip after irradiation.Irradiation was carried out at 308 ± 10 nm for 16 min.

Figure 10 .
Figure 10.Pathways of H4Bip transformation during autoxidation and photooxidation (in vitiligo phototherapy).Author Contributions: Conceptualization, T.A.T. and A.A.B.; methodology, T.A.T. and A.A.B.; validation, T.A.T. and A.A.B.; investigation, Yu.L.V., V.A.B., T.A.T. and A.A.B.; writing-original draft preparation, Yu.L.V., T.A.T. and A.A.B.; editing, T.A.T., Yu.L.V., V.A.B. and A.A.B.; project administration, A.A.B.All authors have read and agreed to the published version of the manuscript.Funding: The research of A.A.B. and T.A.T. was supported by the Russian Science Foundation grant 20-73-10029.The research of Yu.L.V. and V.A.B. was supported by the Ministry of Science and Higher Education of the Russian Federation.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Data are contained within the article.
Bip is prone to autoxidation in the presence of molecular oxygen.The autoxidation starts with an electron transfer from H 4 Bip to O 2 , which is accompanied by tetrahydrobiopterin radical cation (H 4 Bip •+ ) and superoxide radical anion (O 2

Table 1 .
Feasibility of H 4 Bip photooxidation reactions.The Gibbs free energy of the reactions is calculated with the MP2/6-31G(d,p)+ method.COSMO water with a dielectric constant equal to 80 is the solvent.

Table 1 .
Feasibility of H4Bip photooxidation reactions.The Gibbs free energy of the reactions is calculated with the MP2/6-31G(d,p)+ method.COSMO water with a dielectric constant equal to 80 is the solvent.
. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 19 ∆fGº is −58.4 kcal mol −1 when one of the H2Ptr molecules is in the triplet state:

Table 2 .
Calculation of the fraction of oxidized protein (FOP) for HSA under irradiation at 308 nm in the presence and absence of H 4 Bip.