Nickel-catalyzed N-terminal Oxidative Deamination in Peptides Containing Histidine at Position 2 Coupled with Sulfite Oxidation*

Peptides containing histidine at position 2 were observed to undergo spontaneous N-terminal oxidative deamination in aqueous solution in the presence of Ni(II), sulfite, and ambient oxygen. The reaction resulted in the formation of a free carbonyl on the N-terminal α-carbon (α-ketoamide) and was catalytic with respect to nickel. This oxidative deamination was confirmed by13C NMR, 1H NMR, mass spectrometry, and chemical tests. No evidence of modification of histidine was found. It was demonstrated that the nickel-dependent N-terminal oxidative deamination also occurred in His-2 peptides using potassium peroxymonosulfate (oxone) as an oxidant. When oxone was used, oxygen was not required for the deamination to proceed. The results suggest that both nickel-catalyzed reactions (sulfite and oxygen, and oxone) produce an imine intermediate that spontaneously hydrolyzes to form the free carbonyl. These findings may provide a physiologically relevant model for oxidative carbonyl formation in vivo, as well as a useful method for producing a site-specific carbonyl on peptides and proteins.

Excessive exposures to both nickel and sulfite are known to produce serious toxic and carcinogenic effects in animals and humans (1)(2)(3)(4). The generation of potentially harmful radicals from sulfite oxidation by transition metal catalysis has been well established (5). Nickel salts are physiologically redoxinactive, and therefore nickel toxicity in vivo is inferred to involve activation by coordination with peptides and proteins. A variety of catalytic oxidative properties of Ni(II)/peptide complexes have been reported in the literature. These include decarboxylation of the Ni(II)-Gly-Gly-His complex in the presence of molecular oxygen (6), the ability of Ni(II)-histidyl peptide complexes to function as Fenton reaction catalysts (7,8), and the ability of the Ni(II)-Gly-Gly-L-His complex to catalyze the oxidation and cleavage of DNA (9,10) and to promote the formation of intermolecular protein cross-links (11,12). Muller et al. (13) and Liang et al. (14) have recently reported that autooxidation of sulfite catalyzed by the Ni(II)-complexed Lys-Gly-His-amide tripeptide can oxidatively damage DNA. They postulate in situ formation of monoperoxysulfate, a strong oxidant, as an active intermediate in the damaging effect.
We have recently reported on the identification of a unique Ni(II) binding site on hemoglobin that, in the presence of monoperoxysulfate, produces N-terminal oxidative deamination, as well as intramolecular cross-linking, both specific to the ␤-globins (15). In the present study, we have employed small model peptides in order to verify the structural assignment of oxidative deamination, as well as to identify the minimal sequence requirements for reaction susceptibility. Additionally, we have tested a system that substitutes sulfite (SO 3 2Ϫ ) and oxygen (O 2 ) for potassium peroxymonosulfate (oxone). 1 Our findings show clearly that histidine at position 2 is a fundamental requirement for Ni(II)-catalyzed oxidative Nterminal deamination and that sulfite and ambient oxygen can readily substitute for the potent peroxyl oxidant oxone. Furthermore, by contrasting some of the differences between products produced by the sulfite/oxygen and the oxone reactions with Ni(II) peptides, we can exclude the formation of diffusible monoperoxysulfate (HSO 5 Ϫ ) as a mediator in SO 3 2Ϫ /O 2 -promoted deamination.
Sulfite Reaction Standard Conditions-Unless otherwise noted, reaction mixtures contained 30 mM peptide and 5 mM nickel chloride, to which sodium sulfite was added from a 10ϫ stock to a final concentration of 60 mM, all in 0.1 M sodium phosphate, pH 8.2. 300-l reactions were maintained at room temperature for 24 h under ambient air in 16 ϫ 100-mm glass test tubes protected from light. Reactions were quenched by addition of 1: 10 (v/v) of 0.5 M EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at 4°C until analysis by reversed phase chromatography.
Oxone Reaction Standard Conditions-Initial reaction mixtures contained 30 mM peptide and 30 mM nickel chloride in 0.1 M sodium phosphate, pH 8.2, to which oxone, also in 0.1 M sodium phosphate, pH 8.2, was added as a 10ϫ stock to a final concentration of 30 mM. After approximately 5 min at room temperature, the oxone reactions were quenched by the addition of (at 1:10 (v/v)) 0.5 M EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at 4°C until analysis by reversed phase chromatography.
With time, optimized conditions for the oxone reactions were established. Where noted as optimized oxone conditions, the peptides, NiCl 2 , and oxone stocks were made up in 0.2 M sodium bicarbonate, pH 8.2. Additionally, under optimized conditions, the oxone was added in five equal increments 2 min apart, rather than as a single bolus.
Amino Acid Analysis-Peptides were subjected to gas phase hydrolysis at 165°C for 1 h in the presence of HCl containing 1% phenol, using a Savant AP100AminoPrep Hydrolyzer. Amino acids were analyzed using precolumn derivatization with AQC as described previously (16).
Derivatization with 2,4,-Dinitrophenylhydrazine-Derivatizations were performed as described previously (15). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
LC-MS-Electrospray mass spectrometry was performed using a Finnigan Mat LCQ TM ion trap mass spectrometer interfaced with an HP1090M HPLC. The LCQ™ spectrometer was calibrated as per the manufacturer's recommendations, which provide for a specified accuracy of 0.01% for m/z masses of 100 -2000 Da. Peptides were separated at room temperature using a Zorbax 300SB C18 column with a gradient of 1-40% acetonitrile in 0.1% trifluoroacetic acid/water (v/v) in 20 min at 1 ml/min, following an initial 4-min isocratic (1%) hold.
NMR Analysis-1 H and 13 C NMR spectra were collected on a Varian VXR-300S instrument. Peptides were dissolved in either deuterium oxide or deutero-Me 2 SO. Chemical shifts are reported relative to DSS for deuterium oxide samples or tetramethylsilane for deutero-Me 2 SO samples. 1 H chemical shift versus pH studies were performed using a D 2 O-phosphate system. No corrections of pH measurement for D 2 O content were made.

Reaction of the Ni(II)-complexed Tripeptide Ala-His-Ala with
Peroxymonosulfate Produces N-terminal Oxidative Deamination-Reaction of the tripeptide Ala-His-Ala with Ni(II) and oxone under initial standard conditions resulted in Ͼ60% modification of the peptide. The modified peptide eluted as a broad, asymmetric peak several minutes later than the parent peptide peak when analyzed by reversed phase C18 HPLC (Fig. 1, A  and B). The modified peptide displayed significantly increased absorption at 245 nm. Also, the modified peptide displayed a mass of 296.1 Da when analyzed by in-line mass spectrometry. This represents a discrete loss of 1 Da when compared with the parent peptide (296.1 versus 297.1 Da for the parent peptide). MS 2 fragmentation mapped the 1-Da loss to the A 2 , B 2 , and B 3 fragment ions (Fig. 2, A and B). Y-type ions were not detected in either case. The ion of m/z 227, which can be attributed to Y 2 structure, was instead assigned to loss of the C-terminal Ala residue from the AHA tripeptide (17). Ion fragment assignments were confirmed by MS 3 . A significant recovery of the histidine immonium fragment ion was observed (of m/z 110), which further indicated that the histidine residue was unaffected. These findings all suggested that the 1-Da mass loss was located on the the N-terminal alanine, and the histidine residue remained unchanged.
The modified peptide was blocked to Edman protein sequencing, suggesting that a chemical modification had occurred at the N terminus. This finding was corroborated by a lack of reactivity with AQC reagent, which was used to probe for the presence of primary or secondary amines. Amino acid analysis of the modified peptide showed approximately one-half of the yield of alanine relative to histidine that was observed for the parent peptide. Also in contrast to the parent peptide peak, the modified peptide peak reacted quantitatively with DNP, yielding a derivatized peptide with 400 nm absorbance and mass of 476.1 Da (Fig. 3). This represents a mass gain of 180 Da, which is characteristic of a dinitrophenylhydrazone product and therefore indicative of the presence of a free carbonyl on the modified peptide. The presence of a free carbonyl, the apparent loss of free amino group reactivity, and the 1-Da mass decrease all indicated that the modified peptide was oxidatively deaminated at the N terminus, thus resulting in an ␣-ketoamide peptide (Reaction I). 1 H and 13 C NMR analyses of the modified peptide peak were used to further verify this structural assignment. The 1 H spectrum of the (putative) oxidatively deaminated peptide displayed a shift of the N-terminal alanine methyl group downfield from 1.23 to 2.32 ppm along with a resonance splitting change from a doublet to a singlet, consistent with formation of a ketone at the ␣-carbon of Ala-1 ( Table I). The C-1 vinyl proton of the histidine imidazole ring was observed to be significantly deshielded relative to the parent peptide. However, an additional study that examined the effect of pH versus 1 H chemical shift of the histidine C-2 vinyl proton demonstrated no significant change in pK a of the imidazole group following Ni(II)/ oxone oxidation, yielding further evidence that the histidine side chain was not modified in the reaction (Fig. 4). 13 C NMR also indicated the presence of an intact histidine imidazole ring, two amide carbons, one carboxylic acid carbon, and a downfield ketone carbonyl resonance at 196 ppm, all in agreement with oxidative deamination (Table II).
Sodium cyanoborohydride treatment of the modified peptide peak yielded quantitative reduction of the Ni(II)/oxone-oxidized AHA peptide, resulting in a species that eluted earlier in the reversed phase gradient as a doublet of two apparent diastereomers, both exhibiting mass gains of 2 Da (Fig. 1C). These peaks were structurally assigned as diastereomeric al-cohols derived from reduction of the ␣-keto carbonyl. MS 2 fragmentation ( Fig. 2C), as well as 1 H NMR analysis of these peaks, confirmed this assignment.
AQC derivatization of the AHA deamination reaction mixture analyzed by LC-MS yielded evidence of free ammonia released in the reaction. The amount detected was commensurate with the amount of peptide oxidatively modified, showing that deamination was concomitant with the proportional release of free ammonia.
The same oxone reaction on the AHA tripeptide in the absence of added Ni(II) was tested. It was found that when Ni(II) was left out of the reaction, under otherwise identical conditions, about 50% of the peptide was modified. The modified species exhibited an increased absorbance at 250 nm and eluted during the reversed phase HPLC gradient as a sharp, symmetrical peak later than either the parent peptide or the oxidatively deaminated species (Fig. 1D). LC-MS analysis of this peak showed a mass gain of 14 Da over the parent peptide mass (311.1 versus 297.1 Da for the parent). This modified peak was found to be blocked to Edman chemistry sequencing, suggesting that a chemical modification had occurred at the N terminus. In contrast to the oxidatively deaminated AHA peptide peak, this modified peptide peak was unreactive with DNP, suggesting the absence of any free carbonyl group.  Fig. 1C; D, modified peptide from oxone reaction without Ni(II) (oxime). Annotation of fragment ions refers to parent peptide. and B 3 fragment ions (Fig. 2D). From these findings, we postulated that this modification was likely the result of chemical oxidation of the terminal amine nitrogen to an oxime (18). 1 H proton NMR analysis confirmed this assignment. The N-terminal methyl resonance was shifted downfield from 1.23 to 1.85 ppm and resonated as a singlet. Also observed was the appearance of a single, sharp resonance far downfield at 11.86 ppm, which is consistent with an oxime proton.

Reaction of the Ni(II)-complexed Tripeptide Ala-His-Ala with Sulfite and Oxygen Produces Identical N-terminal Oxidative
Deamination-Muller et al. (13) have postulated the in situ formation of HSO 5 Ϫ from SO 3 2Ϫ in the presence of ambient oxygen, Ni(II), and the tripeptide Lys-Gly-His-amide. Although Ni(II) complexes of this sequence motif have been reported to exhibit catalytic properties different from that of histidine at position 2 (7), we decided to test for indications of peroxymonosulfate formation from sulfite autooxidation on a peptide with histidine at position 2.
The tripeptide Ala-His-Ala was incubated with sodium sulfite in the presence of nickel under the standard conditions described above. This treatment was observed to produce modification of Ͼ60% of the peptide. The modified species displayed the same elution time, 1-Da mass loss, and MS 2 fragmentation pattern by LC-MS analysis as the oxidatively deaminated ␣-ketoamide species produced using oxone. Additional chemical tests, including Edman sequencing, DNP, and AQC reactivity, as described above for the modified product of AHA using Ni(II) and oxone (excepting proton NMR analysis), confirmed that an identical oxidatively deaminated AHA peptide was produced using Ni(II), sulfite, and ambient oxygen.
The sulfite reaction with AHA in the absence of added nickel was tested. It was found that in contrast to the oxone reaction, no oxime formation was observed. In fact, when nickel was omitted from the reaction of the AHA tripeptide with sulfite, no detectable modification of any kind was observed.
Reaction of the Ni(II)-complexed Tripeptide Ala-Ala-Ala Shows No Evidence of Oxidative Deamination with Either SO 3 2Ϫ /O 2 or Oxone-We have previously postulated that histidine at position 2 is an essential part of an oxidatively reactive Ni(II) binding site on the ␤-chain of human hemoglobin (15). Therefore, as a comparison with the AHA peptide, we examined the reactivity of the tripeptide AAA. Reaction of the AAA tripeptide with Ni(II) and oxone under standard conditions also resulted in Ͼ60% modification of the peptide. This modified peptide eluted primarily as a sharp, symmetrical peak several minutes later than the unmodified parent peptide peak when analyzed by reversed phase C18 HPLC (Fig. 5, A and B). The modified peptide displayed increased absorption at 250 nm and a discrete mass gain of 14 Da when analyzed by LC-MS compared with the parent peptide (245.1 versus 231.1 Da for the parent peptide.) This species was found to be blocked to Edman sequencing. MS 2 fragmentation also indicated that the modification was located on the N-terminal amino acid. Based on these findings, we concluded that this modification was an oxime on the N-terminal nitrogen. When nickel was omitted from the oxone reaction with the Ala-Ala-Ala tripeptide, an identical product was observed (Fig. 5C). Therefore, oxime formation appears to be entirely non-nickel-dependent.
Reaction of the tripeptide Ala-Ala-Ala with Ni(II) and sodium sulfite under standard (SO 3 2Ϫ ) conditions produced no detectable modification of any kind. This finding suggests that in the case of the Ala-Ala-Ala tripeptide, no peroxymonosulfate-like species is generated by that peptide in the presence of Ni(II), SO 3 2Ϫ , and ambient oxygen. Histidine at position 2 therefore appears to be required for both susceptibility to oxidative deamination, as well as the apparent ability to generate a peroxymonosulfate-like species from sulfite and oxygen.
Sequence  tions of Ni(II) and a series of small peptides with either SO 3 2Ϫ / O 2 or oxone as the oxidant. We examined the reactions for any evidence of modification of the parent peptide. The results of these experiments are shown in Table III.
Almost all of the peptides with histidine at position 2 demonstrated significant susceptibility to oxidative deamination under both sets of conditions, whereas none of the peptides lacking histidine at position 2 demonstrated any oxidative deamination under either condition. It should be noted that in the cases of both AHA and GHG, a significant amount of the deaminated product from reaction at 30 mM peptide was found to be in the form of apparently diastereomeric pairs of relatively stable dimers (exhibiting a mass of 592 Da in the case of AHA peptide). These dimers were not observed when the SO 3 2Ϫ / O 2 reaction was performed at a peptide concentration of 3 mM, indicating that their formation was (peptide) concentration-dependent. Further characterization of the dimer by 1 H NMR indicated it to be a product of aldol condensation of two molecules of ␣-ketoamide (data not shown). These dimers were not observed when oxone was used as the oxidant.
Unlike Ala-His, the dipeptides carnosine (␤-alanyl-histidine) and homocarnosine (␥-amino butyryl-histidine) were found to be nonreactive to Ni(II)/SO 3 2Ϫ /O 2 -promoted deamination. These results suggest that only ␣-amines are susceptible to this pathway of deamination. Interestingly, these peptides showed very little oxime formation in the reaction with oxone.
Peptides with proline as the first residue also produced noticeably different results. N-terminal proline, being a secondary amine, is not likely to be as susceptible to the oxidative release of ammonia as other amino acids. No released ammonia in either reaction with PHA was detected, and in both cases, a modified peptide that was formed demonstrated a loss of 2 Da (321 versus 323 Da for the parent peptide). The modified peptide was unreactive with AQC, indicating that the secondary amine was no longer present. This result would be consistent with formation of a cyclic imine species that was stable against spontaneous hydrolysis.
Peptides GGH and KGH-amide with a histidine residue in position 3 were also tested. Under similar conditions, these peptides have been shown by Muller et al. (13) and others (12,14,19) to produce radicals capable of causing DNA damage and protein cross-linking. No deamination of either the GGH or the KGH-amide peptides were detected in reactions with Ni(II)/ SO 3 2Ϫ /O 2 . The KGH-amide peptide was modified in the reaction to an earlier eluting product exhibiting a mass gain of 80 Da, consistent with the displacement of one proton by an SO 3 H group. This product was not further characterized. In the reactions with oxone, decarboxylation of GGH and other peptide degradation of both peptides were observed, none of which appeared to include deamination. By contrast, the tripeptide KHG-amide (bursin) appeared to readily N-terminally deaminate with both Ni(II)/SO 3 2Ϫ /O 2 and Ni(II)/oxone. However, the ketoamide product appeared to spontaneously form an intramolecular Schiff's base with the ⑀-amine of the lysyl side chain (Reaction II). This was confirmed by LC-MS, Edman sequencing, and reaction with DNP. Under these conditions, no evidence of ⑀-amino side chain deamination was found.
Peptides with methionine and tyrosine as the N-terminal residues also appeared to present exceptions to susceptibility to oxidative deamination. The lack of reactivity of MH using sulfite is possibly due to sulfoxide formation acting as a "sink" for oxidative modification, although no substantial difference in the amount of methionine sulfoxide formed was seen relative to ML with either oxidant (20,21). It is also conceivable that other chemical and electronic properties of the sulfur atom of the methionine side chain could affect the reaction pathway. The lack of deamination of the dipeptide YH is less clear. Stable tyrosine radicals have been reported in proteins (22,23). However, no evidence of dityrosine, a known product of oxidative degradation of tyrosine involved in protein cross-linking, was detected (12,22), and no evidence of tyrosine radical was found by EPR or found to be trappable by the method of Fenwick and English (24). However, with an increased excess of sulfite, we noticed significant accumulation of a product of the YH reac-REACTION II REACTION III tion, which exhibited a mass of 354 Da (gain of 36 Da). Under standard conditions, except using 4:1 ratio of sulfite over peptide, the reaction resulted in a 30% yield of this modification. Mass gain, MS 2 fragmentation, and Edman protein sequencing were all consistent with C-terminal decarboxylation followed by the attachment of the SO 3 H group forming a sulfo-peptide (Reaction III). We believe that such a product would most likely be formed through a radical type of reaction, potentially promoted by the tyrosine side chain (25). Analogous products were detectable with other first residue side chains in dipeptides containing His-2, but with yields at least 1 order of magnitude lower. The YH sulfo-peptide product was not observed when Ni(II) was omitted from the reaction. The Ni(II)/oxone reaction with YH produced a small but detectable yield of oxidative deamination. Additionally, a significant amount of other uncharacterized degradation occurred including formation of a yellow precipitate.
In all cases, oxone produced a significant yield of oxime in peptides lacking histidine at position 2. This finding provided us with a means to look for evidence of the generation of a diffusible peroxymonosulfate species from peptide/Ni(II) reac-tions with sulfite. This could be tested by including the AAA tripeptide in reactions with His-2-containing peptides and monitoring for oxime formation. These conditions resulted in substantial deamination of the susceptible peptides, as was seen with oxone addition. No trace of oxime formation was detectable on the AAA tripeptide when it was combined with the LH dipeptide during standard Ni(II)/SO 3 2Ϫ /O 2 conditions. Therefore, it can be inferred that the oxidative deamination reaction did not generate any appreciable quantities of diffusible peroxymonosulfate. However, in the case of all His-2 peptides, an in situ formation of a peroxymonosulfate-like species, which is either constrained from diffusing or consumed in a subsequent reaction too quickly to do so, is plausible.

Nickel Titration Studies of LH Dipeptide Reacted with Ni(II) and Either SO 3 2Ϫ /O 2 or
Oxone-A nickel titration study of the SO 3 2Ϫ /O 2 reaction was conducted using the dipeptide LH under standard conditions. The results showed that in the absence of nickel, no oxidative deamination occurred. At a Ni(II) to peptide ratio of 1:10, about 40% of the parent peptide was deami-   nated (i.e. 4 equivalents of deaminated peptide per 1 equivalent of Ni(II)), indicating a catalytic turnover of 4. Using a 1:10 ratio of nickel, and 4 equivalents of sulfite, the catalytic turnover of Ni(II) increased to about 8.5. Increasing the Ni(II)/peptide ratio above 1:5 did not have a significant effect on the yield of deamination (Fig. 6). Therefore, a 1:5 ratio of Ni(II):peptide was selected as an optimal for the standard deamination reaction with sulfite.
A similar Ni(II) titration study using the LH dipeptide was conducted with oxone. In contrast to the SO 3 2Ϫ /O 2 system, under initial conditions, maximal oxidative deamination required nearly 1:1 Ni(II):peptide when oxone was used. However, under optimized conditions for the oxone reaction, the oxone was added to the reaction in five equal increments, rather than as a single bolus. In this case, 30% deamination was achieved using approximately 1:5 Ni:peptide (catalytic turnover of 1.5), suggesting that redox cycling of Ni(II) can be achieved in the oxone reaction as well (Fig. 6). Chromatograms of this "optimized" oxone reaction, along with a standard condition sulfite reaction, both with the LH dipeptide at 1:5 Ni(II):peptide, are shown in Fig. 7. It can be seen from these that the sulfite reaction results in significantly decreased degradative side products compared with oxone. It appeared that when nickel was limiting, oxime formation was a potentially competing side reaction. We therefore purified the LH oxime species and rereacted it with Ni(II) and oxone under standard conditions. No evidence of oxidative deamination was observed, although significant heterogeneous, uncharacterized peptide degradation occurred. This finding suggested that oxime formation may be a competitive and kinetically relevant "dead-end" side reaction when Ni(II) is limited during reaction with oxone.
Reaction Kinetics for Oxidative Deamination of LH Dipeptide Reacted with Ni(II) and Either SO 3 2Ϫ /O 2 or Oxone-Studies were conducted to examine the kinetics of oxidative deamination of the LH dipeptide comparing the use of either SO 3 2Ϫ /O 2 or oxone (with sequential addition) as oxidants (Fig. 8). As can be seen, the oxidative deamination using oxone as the oxidant was completed within (at most) several minutes of initiation. By contrast, the reaction using SO 3 2Ϫ and ambient oxygen displayed a distinctly lower initial rate of deamination, which appeared to be relatively constant during the first 40 min of reaction. No further increase in deamination was observed beyond 6 h (up to 1 week). When this reaction was repeated under an oxygen-enriched head space (Ͼ95% O 2 at approximately 2 atm), no difference in the rate of peptide modification was observed. This finding suggested that oxygen diffusion is not rate-limiting under the standard conditions with sulfite. However, when oxygen was strictly excluded from the sulfite reaction (under argon), no peptide modification was observed. By contrast, exclusion of oxygen from the oxone reaction did not inhibit oxidative deamination.

Quenching and Inhibition Studies of Deamination of LH Dipeptide Reacted with Ni(II) and Either Sulfite or Oxone-
Further studies were conducted to examine the effect of the addition of various compounds on the yields of oxidative modification in the LH/Ni(II) reactions using either sulfite or oxone. These additives included known chelating agents, radical scavengers, and different anions. The results of these experiments tabulated as percentage of inhibition of LH deamination under the respective standard conditions are listed in Table IV. The lack of inhibition observed in the presence of ethanol, methanol, tert-butyl alcohol, or mannitol argues strongly against the involvement of diffusible SO 4 . or OH ⅐ radicals me-  diating the oxidative deamination in either reaction (2,26). The inability of catalase to inhibit either reaction suggests that it is unlikely that oxidative deamination in either case proceeds through formation of diffusible H 2 O 2 followed by a Fenton-type reaction, as in a mechanistic scenario for oxidative protein carbonyl formation proposed by Stadtman (27). However, no test was conducted to determine whether this enzyme was inactivated by the reaction conditions, as has been reported for sulfite inactivation of several copper-dependent monooxygenases (28). The complete inhibition of oxidative deamination by the metal chelating agent EDTA would suggest that sequestration of nickel away from the peptide is the mode of action by which it precludes the reactions. The mechanism for inhibition exhibited by thiourea is less clear, but the differential effects of CN Ϫ and HCO 3 Ϫ on reactions using SO 3 2Ϫ /O 2 versus using

HSO 5
Ϫ begins to suggest that these may act by perturbing the coordination structure of the reactive nickel complexes (29). Such differential effects also begin to suggest that the reactive complex that produces an actively oxidizing species from SO 3 2Ϫ may differ from the one involved in the reaction with preformed HSO 5 Ϫ (oxone). Comparative Sulfite and Oxone Titration Studies with Ni(II)complexed LH Dipeptide-Titration studies under standard conditions using oxone as the oxidant showed that approximately 1:1 oxone:peptide was sufficient to approach maximal deamination of the LH dipeptide (Fig. 9). It was also observed that Ն2:1 oxone:peptide resulted in significant, heterogeneous, uncharacterized degradation of the peptide (much worse than the 1:1 shown in Fig. 7). By contrast, titration with SO 3 2Ϫ under standard conditions reproducibly showed that significantly more than 1 equivalent of SO 3 2Ϫ to peptide was required to approach maximal deamination. With 3 equivalents of sulfite, more than 60% of the peptide was oxidatively deaminated, with less than 20% of degradative side-products observed. pH

Effect Studies of LH Dipeptide Reacted with Ni(II) and Either Sulfite/O 2 or Oxone Show High Relative Yields of Oxidative Deamination throughout a Physiological pH Range-
Reactions with the LH dipeptide/Ni(II) system using oxone or sulfite/O 2 were examined for effects of reaction pH, the results of which are shown in Fig. 10. Although the oxone reaction showed inhibition below pH 6.5, both reactions displayed a high relative yield of oxidative deamination over the physiologically relevant pH range of 6.5-8.5. This finding lends support to the possibility that this model system for oxidative peptide modification may be relevant to mechanisms of nickel and sulfite toxicities in vivo (1, 3).
Conclusions-We have shown that the reaction of peptides containing histidine at position 2 with Ni(II) and oxone produces oxidative deamination of the peptides with a minimum of other significant modifications of the peptide, and we found no evidence of modification of the histidine residue (up to 1 equivalent of oxone, above which other degradative modifications occur). When Ni(II) was omitted from the reaction, oxime formation on the N-terminal nitrogen was produced instead. In peptides lacking histidine at position 2, only oxime formation was observed, regardless of whether Ni(II) was present or not.
The identical oxidative deamination of peptides containing His-2 can be produced by using Ni(II), sulfite, and ambient oxygen. A substoichiometric amount of nickel can serve to deaminate the peptides in high yields, indicative of redox cycling of Ni(II) in the reaction. However, when either nickel, sulfite, or oxygen was excluded, no oxidative deamination or any other modification was observed. No oxidative deamination was observed in any peptides lacking histidine at position 2. Significant modifications other than deamination were observed in reactions with both GGH and KGH-amide peptides. These peptides have been reported to promote protein cross- linking and DNA damage under similar conditions. Additionally, the GGH peptide is known to undergo oxidative degradation, including decarboxylation in the presence of ambient oxygen or other oxidizers (6,14,30). These results, along with our finding of oxidative deamination only on ␣-carbon amines in His-2 peptides, indicate a unique structural specificity for a sequence motif that was first discovered in the ␤-chain of human hemoglobin (15).
The finding that both sulfite/O 2 and oxone produce an identical ␣-ketoamide suggests that both pathways may share a common reactive intermediate. The Ni(II)-catalyzed formation of peroxymonosulfate from sulfite and oxygen would appear to be consistent with these results (13). However, co-incubation of peptides lacking histidine at position 2 exposed to the Ni(II), sulfite, and oxygen system showed no evidence of oxime formation, suggesting that no diffusible peroxymonosulfate is produced during the deamination reaction. Additional quenching studies using well characterized radical scavengers appear to rule out diffusible OH ⅐ or SO 4 . as mediating any of the oxidative deamination observed (2,26). We postulate that the reactions of Ni(II) complexed His-2 peptides with either oxone or an in situ formed, nondiffusing, peroxymonosulfate-like species generated catalytically by sulfite autooxidation produce a common imine intermediate. All of our results would be consistent with a pathway involving imine formation at the N terminus, followed by spontaneous hydrolysis (with the exception of proline, which apparently forms a stable cyclic imine). The finding of ammonia release in all cases of oxidative deamination is consistent with such a pathway. This postulated pathway is shown in Fig. 11. Imine formation in oxidative deamination has been postulated by Stadtman (27) to be preceded by radical formation on the carbon adjacent to the departing amine group. If such is the case for Ni(II)-catalyzed N-terminal oxidative deamination, the radical appears inaccessible to quenching by common radical scavengers, i.e. a type of "caged" radical. Results with YH dipeptide suggest that the (tyrosyl) side chain can redirect the outcome of the reaction of such a complexed radical.
These findings are of interest from several perspectives. They provide a defined, potentially physiologically relevant model system with which to study the mechanism of a metalcatalyzed protein carbonyl formation, a modification known to be a frequent consequence of oxidative damage to proteins in vivo (31,32). Such a defined system may provide an opportunity for further elucidating mechanisms underlying the known toxicities of sulfite, a ubiquitous environmental contaminant (2,3). Additionally, our findings point toward a facile method for the intentional introduction of a carbonyl "handle" into natural and engineered proteins and peptides, under conditions that produce relatively few undesirable side reactions, a technology of significant potential utility (33,34).