(Received for publication, June 26, 1995; and in revised form, November 2, 1995)
From the
During exposure to ozone, the methionine and aromatic amino acid
residues of Escherichia coli glutamine synthetase (GS) and
bovine serum albumin (BSA) are oxidized rapidly in the order Met >
Trp > Tyr His > Phe. The loss of His is matched by a nearly
equivalent formation of aspartate or of a derivative that is converted
to aspartic acid upon acid hydrolysis. Conversion of His to aspartate
was confirmed by showing that the oxidation of E. coli protein
in which all His residues were uniformly labeled with
C
gave rise to
C-labeled aspartic acid in 80% yield and also
by the demonstration that His residues in the tripeptides Ala-His-Ala
or Ala-Ala-His gave rise to nearly stoichiometric amounts of aspartic
acid whereas oxidation of His-Ala-Ala yielded only 36% aspartate. The
oxidation of BSA and GS led to formation, respectively, of 11 and 3.3
eq of carbonyl groups and 0.5 and 0.3 eq of quinoprotein per subunit.
Although BSA and GS contain nearly identical amounts of each kind of
aromatic amino acid residues, oxidation of these residues in BSA was
about 1.5-2.0 times faster than in GS indicating that the
susceptibility to oxidation is dependent on the primary, secondary,
tertiary, and quaternary structure of the protein.
Ozone is one of the most toxic pollutants in the atmosphere. Brief exposure to concentrations of ozone below 1 ppm leads to damage of the lung, especially to regional damage of bronchiolar ciliated cells and alveolar epithelial type I cells. Prolonged exposure leads to inhibition of ciliagenesis and type 2 cell maturation, to inflammation, fibrosis, enhanced collagen synthesis, and to increased sensitivity to bacterial infection (for review, see Mehlman and Borek(1) , Menzel(2) , and Stokinger(3) ). Pryor (4) has argued that ozone reacts within the alveolar epithelial type I cells so rapidly that it cannot cross the lung-lining fluid layer except in patchy areas where the lower airways are virtually uncovered. This accounts for the observation that tissue damage by ozone is largely restricted to the lung. Nevertheless, exposure of rats to ozone has been shown to elicit a number of extrapulmonary effects, including a decrease in visual acuity(5) , defects in the desaturation of oxyhemoglobin in skin capillaries(6) , changes in cardiac protein metabolism(7) , increased lipid peroxidation in heart and brain tissue, and the elevation of peroxide scavengers in these tissues(8) .
Because of its pronounced cytotoxicity, recent
studies have focused on the fact that ozone can give rise to a number
of other reactive oxygen species including OH,
HO
, O
,
RCOO
, O
, and singlet
oxygen(9, 10, 11, 12, 13, 14, 15) .
In some circumstances, these secondary reactive oxygen species might be
more damaging than ozone itself. Thus, the reaction of ozone with
polyunsaturated fatty acids in membrane lipids leads to the generation
of relatively stable lipid peroxides, which can subsequently give rise
to tissue-damaging alkoxy radicals and alkyl
radicals(12, 15, 16) . Lipid-independent
mechanisms must also be considered since ozone reacts directly with
some amino acids(17, 18) , with proteins (see (1) for review), and with nucleic
acids(19, 20) . In the case of free amino acids and
amino acid residues in proteins, cysteine, methionine, tryptophan,
phenylalanine, and histidine, residues are particularly sensitive to
oxidation by ozone; other amino acids are fairly resistant to
ozone(17, 21) . Kynurenine and N-formylkynurenine have been identified as major products of
free tryptophan and of tryptophan residues in proteins(22) .
Cysteic acid and cystine are major products of cysteine
oxidation(2, 22, 23) . Methionine sulfoxide
is a major product of methionine oxidation(17) .
Dihydroxyphenylalanine was produced in low yields during the oxidation
of tyrosine by ozone. Ammonia and an amino acid tentatively identified
as proline are products of histidine oxidation at pH 4.6(17) .
The rates at which the aromatic and sulfur amino acid residues of
proteins are oxidized by ozone varies widely from one protein to
another, suggesting that the susceptibility to oxidation is dependent
upon the size and amino acid composition as well as the secondary and
tertiary structure. In the present study, we compare the ability of
ozone to oxidize amino acid residues in Escherichia coli glutamine synthetase (GS) ()and bovine serum albumin
(BSA). These two proteins are similar with respect to subunit size (50 versus 67 kDa), they contain nearly equal numbers of Trp, Tyr,
His, and Phe residues per subunit, and x-ray crystallographic data are
available for GS (24) and human serum albumin (25) ,
which is highly homologous to BSA. The GS was selected also because the
results could be compared with those derived from extensive studies on
the oxidation of the enzyme by metal-catalyzed oxidation (MCO) systems.
In addition, we investigated the ozone-mediated oxidation of a series
of tripeptides, each of which contained two alanine residues and one
histidine residue that was present in either the N-terminal, middle, or
C-terminal position.
From the data in Fig. 1and other data not shown, it is evident that the modification of a given kind of aromatic amino acid residue is a nearly linear function of time until at least 60 to 90% of the residues have been oxidized. Also, as shown in Table 1, the specific rate constant k = (number of residues modified per min)/(total number of residues present in the protein) for each aromatic amino acid is about 1.5-2.0 times higher for residues in BSA than in GS. Since GS and BSA contain nearly equal amounts of each of the aromatic amino acids (Table 1), this difference in susceptibility to oxidation by ozone likely reflects differences in the primary, secondary, tertiary, or quaternary structures. In this regard, it is noteworthy that each of the 12 identical subunits of GS are arranged in two superimposed hexaganol arrays, whereas BSA exists as a monomer.
Figure 1: Ozone-induced changes in the number of tyrosine, histidine, and aspartic acid residues of BSA and GS. Mixtures (2.5 ml) containing 10 mM potassium phosphate buffer, pH 7.4, and 0.75 mg/ml BSA (closed symbols) or GS (open symbols) were bubbled with an ozone/oxygen mixture yielding 545 nmol of ozone/min. At times indicated, aliquots were removed and the amino acid composition of the proteins was determined. The time-dependent changes in the number of tyrosine (triangles) and histidine (squares) residues per subunit is indicated in the lower half of the figure. The time-dependent increase in the number of aspartic acid residues in BSA (closed circles) and GS (open circles) is indicated in the upper portion of the figure.
To confirm that ozone promotes conversion
of histidine residues to aspartic acid or to a derivative that yields
aspartic acid upon acid hydrolysis, a protein preparation in which all
of the histidine residues were uniformly labeled with C
was prepared by growing a histidine-requiring auxotrope of E. coli on a medium containing uniformly labeled
[
C]histidine as the sole histidine source. The
[
C]histidine-labeled protein fraction from
cell-free extracts of the organism was then exposed to ozone under our
standard conditions. As shown in Fig. 2, the treatment with
ozone led to a nearly linear, time-dependent decrease in the amount of
C-labeled histidine and was accompanied by a nearly
stoichiometric (approximately 80%) increase in the amount of
C-labeled aspartate that was present following acid
hydrolysis of the ozone-treated protein. Conversion of histidine
residues to aspartate was confirmed also by the demonstration that the
histidine residue in a tripeptide containing one His residue and two
Ala residues was converted to aspartic acid following exposure to ozone
and acid hydrolysis.
Figure 2:
Ozone-elicited conversion of
[C]histidine residues in E. coli protein to [
C]aspartic acid. 4.0 mg of
histidine-labeled protein from E. coli containing 1,032,000
cpm/mg in a total volume of 2.2 ml was bubbled with an ozone-oxygen
mixture at a rate corresponding to 545 nmol/min. At the times
indicated, 0.1-ml aliquots were removed, hydrolyzed in 6 N HCl, and amino acids in the hydrolysate were separated by HPLC of
their OPA derivatives. The histidine and aspartic acid fractions were
collected, and the
C content was determined by the
scintillation counting technique. For plotting, the radioactivity of
the aspartic acid fraction was multiplied by 1.5 to compensate for the
fact that 2 of the 6 carbon atoms of histidine are lost during its
conversion to aspartic acid. The dashed line represents the
expected relationship if all of the histidine that disappeared was
recovered as aspartic acid. The inset shows how the amount of
histidine declines with time of ozone
exposure.
Figure 3: Effect of histidine location in tripeptides on its oxidation. Tripeptides, 1.6 mM in 1.3 ml of 10 mM potassium phosphate buffer, pH 7.4, were treated with ozone at a rate of 242 nmol per min. At the times indicated, 0.01-ml aliquots were removed, and amino acid composition was determined as described under ``Experimental Procedures.'' Symbols are as follows: squares, HAA; circles, AAH; triangles, AHA. A, broken lines and open symbols refer to the amount of histidine residue present at the times indicated. Solid lines and closed symbols refer to the amounts of aspartic acid formed. B, relationship between the amount of histidine residues lost and the amount of aspartic acid formed.
The fact that a histidine residue in the internal position of the peptide is stoichiometrically recovered as aspartic acid explains why the histidine residues in BSA and GS are converted almost entirely to aspartic acid. In contrast, aspartic acid accounts for only 32% of the products formed from a histidine residue occupying the N-terminal position of the peptide. An examination of the ultraviolet absorption spectra of the peptides shows that ozone treatment leads, in the case of HAA, to products possessing much higher absorption in the range of 360 nm than products obtained from either AHA or AAH (Fig. 4). Finally, it appears significant that little or no carbonyl compounds could be detected among the products obtained from any of the histidine-containing tripeptides.
Figure 4: Time-dependent changes in spectrum of tripeptides during ozone exposure. Conditions were as in Fig. 3. At the times indicated, 0.1 ml of reaction mixture was diluted to 1.0 ml and the spectrum was recorded. Curves 1-7 counting from the bottom up refer to spectra taken at 0, 2, 5, 10, 15, 20, and 30 min, respectively.
Figure 5:
Effect of ozone on histidine content and
catalytic activity of glutamine synthetase. GS, 0.75 mg/ml in 2.5 ml of
10 mM potassium phosphate buffer, pH 7.4, was treated with
ozone, 545 nmol/min, as described in Fig. 1. At the times
indicated, aliquots were assayed for histidine content of the protein (circles) and GS -glutamyltransferase activity (squares). The inset illustrates the relationship
between the loss of histidine residues and the loss of GS activity,
expressed as percentages of the amount originally
present.
Figure 6: Ozone-mediated generation of carbonyl groups in GS and BSA. The conditions were as described in Fig. 1. At the times indicated, 0.1 ml of reaction mixtures was assayed for carbonyl content.
Figure 7: Relationship between tyrosine modification and quinoprotein formed. Mixtures (2.5 ml) containing 0.75 mg/ml BSA (circles) or GS (triangles) were bubbled with ozone at a rate of 500 nmol/min. At the times indicated, aliquots were assayed for tyrosine (open symbols) or quinoproteins (closed symbols) as described under ``Experimental Procedures.'' The inset is a plot of quinoidal substances (+Q) formed versus the amount of tyrosine (Tyr) lost, both expressed as nanomoles of residues/nmol of protein subunit.
The demonstration that several kinds of amino acid residues in BSA are more rapidly oxidized by ozone than they are in GS confirms results of earlier workers (21) showing that the sensitivity of a given kind of amino acid residue to oxidation by ozone varies from one protein to another. However, the conclusion that this variability is due to differences in the primary, secondary, tertiary, and quaternary structures of the proteins is strengthened by the results presented here, because the subunits of BSA and GS are of comparable size and contain nearly equal numbers of each of the aromatic amino acids.
Three observations support the conclusion that the histidine
residues in protein and peptides are converted to aspartyl residues or
to derivatives that are converted to aspartic acid upon acid
hydrolysis. 1) The ozone-induced loss of histidine residues in BSA and
GS is accompanied by a nearly stoichiometric increase in the amount of
aspartic acid present in acid hydrolysis of the ozone-treated proteins.
2) The histidine lost during oxidation of the tripeptide, Ala-His-Ala,
is recovered in acid hydrolysates as aspartic acid. 3) When proteins in
which all histidine residues are uniformly labeled with C
are treated with ozone, the labeled histidine which is lost is almost
quantitatively (80%) recovered in acid hydrolysates as
C-labeled aspartic acid.
Of particular interest is the
finding that the ozone-dependent loss of GS activity is directly
proportional to the total number of histidine residues that are
modified (Fig. 5), over a range of histidine residues
representing 75% of all histidine residues in the protein. This is in
sharp contrast to the fact that, when GS is exposed to the
ascorbate/FeIII/O MCO system, the loss of activity is
correlated with the loss of a single histidine residue
(His-269)(36, 38) . Thus, the inactivation of GS by
ozone involves a more or less random attack of histidine residues;
His-269 is no more susceptible to ozone attack than at least 10 other
histidine residues in the molecule (and possibly even less so if
oxidation of any of the other residues causes any loss of catalytic
activity).
In earlier studies, Farber and Levine (37) presented evidence indicating that in the oxidation of GS
by the ascorbate/FeIII/O MCO system His-269 is converted to
an asparaginyl residue, which upon acid hydrolysis would be converted
to aspartic acid. In the meantime, prompted by the report of Uchida et al.(47) that the histidine moiety of N-benzoylhistidine is converted to 2-oxohistidine by the
CuII/H
O
/MCO system, Levine and co-workers (
)reinvestigated the metal-catalyzed oxidation of GS using
more advanced technology and have confirmed that the oxidation of
His-269 does indeed give rise to 2-oxohistidine, which under some
conditions can be converted to aspartic acid by acid hydrolysis.
Accordingly, we suspected that the oxidation of histidine residues by
ozone would also yield 2-oxohistidine as the primary product. Thus, if
2-oxohistidine is formed, it can only be a transitory intermediate that
is rapidly converted further to asparagine or aspartate.