Mechanisms of Protection of Catalase by NADPH
KINETICS AND STOICHIOMETRY*
Henry N.
Kirkman
,
Michela
Rolfo§,
Anna M.
Ferraris§, and
Gian
F.
Gaetani§
From the
Department of Pediatrics, University of
North Carolina, Chapel Hill, North Carolina 27599-7487 and the
§ Division of Hematological Oncology, Istituto Nazionale per
la Ricerca sul Cancro and Dipartimento di Oncologia Clinica e
Sperimentale, University of Genoa, Viale Benedetto XV, 10, 16132 Genoa, Italy
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ABSTRACT |
NADPH is known to be tightly bound to mammalian
catalase and to offset the ability of the substrate of catalase
(H2O2) to convert the enzyme to an
inactive state (compound II). In the process, the bound NADPH becomes
NADP+ and is replaced by another molecule of NADPH. This
protection is believed to occur through electron tunneling between
NADPH on the surface of the catalase and the heme group within the
enzyme. The present study provided additional support for the concept of an intermediate state of catalase, through which NADPH serves to
prevent the formation (rather than increase the removal) of compound
II. In contrast, the superoxide radical seemed to bypass the
intermediate state since NADPH had very little ability to prevent the
superoxide radical from converting catalase to compound II. Moreover,
the rate of NADPH oxidation was several times the rate of compound II
formation (in the absence of NADPH) under a variety of conditions. Very
little NADPH oxidation occurred when NADPH was exposed to catalase,
H2O2, or the superoxide radical separately.
That the ratio exceeds 1 suggests that NADPH may protect catalase from
oxidative damage through actions broader than merely preventing the
formation of compound II.
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INTRODUCTION |
Interest in the disposal of reactive oxygen species stems from the
growing evidence that these molecules are active or participating agents in mutagenesis and aging and in the cellular damage from a wide
variety of environmental and endogenous stresses (1-3). One of the
more highly studied cells under oxidative stress is the human
erythrocyte with, and without, a genetic impairment in maintaining NADP
in the reduced state (NADPH) (4-10). The impairment results from the
most common of the potentially lethal human enzyme defects, that of
glucose-6-phosphate dehydrogenase. The susceptibility of such cells to
oxidative stress was initially attributed to the need for NADPH to
remove hydrogen peroxide (H2O2) via glutathione reductase and glutathione peroxidase (4). Attempts to identify a
soluble protein that was binding NADPH within human erythrocytes led to
the discovery that one molecule of NADPH is tightly bound to each of
the four subunits of catalase
(H2O2:H2O2
oxidoreductase, EC 1.11.1.6) of mammals (11). Studies of purified
catalase revealed that NADPH effectively protects catalase against
H2O2 at physiologically realistic
concentrations of both NADPH and H2O2 (12).
These and other findings (13) with highly purified catalase confirmed
studies that led earlier investigators to notice the same effect in
hemolysates (14). This action of NADPH solved a decades-old puzzle as
to the nature of reducing equivalents that serve to keep catalase
active in vivo. Later studies revealed that, within human
erythrocytes, the role of NADPH in keeping catalase active was more
important than the role of NADPH in the glutathione
reductase/peroxidase pathway (7-10). Evidence has been presented that
NADPH protects catalase by preventing and reversing the formation of an
inactive form of catalase, compound II, which differs within the heme
group from active catalase (12). Work in over five laboratories has
provided information on how NADPH could accomplish this task (12,
15-20). Among the difficulties in explaining the protection of
catalase by NADPH is that NADPH is bound on the surface of catalase,
some 20 Å from the heme, that the channel to the heme is too narrow to
accommodate NADPH, and that conversion of compound II back to native
catalase is a one-electron reduction step, whereas NADPH is
traditionally regarded as a two-electron reducing substance. The
present study provides kinetic and stoichiometric observations on the
mechanism of this action by NADPH and suggests how a current model of
this action will need to be revised to accommodate these new findings.
An understanding of the experiments that follow requires knowledge of
the terminology and pathways for the interconversion of the various
forms of catalase. Fig. 1 consists of the
additions of Lardinois (21) to the traditional scheme (22) for those interconversions. The role of NADPH in the scheme is described under
the "Discussion." Ferricatalase has a protoporphyrin IX-iron(III) complex as its active site and is the native, free, or resting state of
the enzyme. Compound I, which is the other active form of catalase,
contains an atom of oxygen gained from step 1 (see Fig. 1 for
steps 1-9), leaving the protoporphyrin-iron group at 2 oxidation equivalents above that of ferricatalase (19). The overall
conversion of H2O2 to H2O and
O2 requires that the enzyme alternate between being
ferricatalase and compound I. Compounds II and III are two inactive
forms of catalase that can arise from exposure of catalase to either
H2O2 (22) or O
2 (19). Compound II
arises by one-electron reduction of compound I and is considered to be
an iron(IV) oxo-ligated porphyrin (23). The one-electron reduction can
result from certain reducing substances of relatively small molecular
size, such as ferrocyanide, or from a poorly understood "endogenous
donor" within the structure of catalase (17, 22). Compound III, also
called oxycatalase, is regarded as having similarity to the oxy
compounds of myoglobin and hemoglobin (21). Although the rate constant
for step 2 is much higher than that for step 9, ethanol can be added at
a concentration greatly exceeding the steady-state concentration of
H2O2 that is generated by glucose oxidase (22).
The resulting severe reduction in concentration of compound I has been
used to demonstrate that compound I is a precursor to compound II (22).
Compound III can arise from the action of H2O2
on compound II or from the action of O
2 on ferricatalase (Fig.
1) (21). Each state of catalase has its own absorption spectrum,
although no single wavelength provides a measurement of just one of the
four states. Compounds II and III revert spontaneously to an active
form of catalase when H2O2 and O
2 are
no longer present. Continual presence of some of the catalase as
compound II or compound III, however, leads gradually to irreversible
inactivation of the enzyme through step 8 (21).

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Fig. 1.
Reactions and interconversions of the four
states of mammalian catalase. Compounds II and III are the
inactive states of catalase. DH represents the endogenous
donor.
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EXPERIMENTAL PROCEDURES |
All incubations were with commercially available, extensively
purified enzymes. Sigma was the source of the manganese superoxide dismutase from Escherichia coli. As with the studies of Kono
and Fridovich (23), manganese superoxide dismutase was used for most of
this study because, unlike Cu,Zn superoxide dismutase, it is not
inactivated by H2O2. Roche Molecular
Biochemicals (Mannheim, Germany) was the source of catalase from bovine
liver, xanthine oxidase from bovine milk, Cu,Zn superoxide dismutase
from bovine erythrocytes, glucose oxidase from Apergillus
niger, and glucose-6-phosphate dehydrogenase from yeast. The
buffer used for most of this study was 0.1 mM EDTA, 50 mM
Na2HPO4-KH2PO4 buffer,
pH 7.4 (Na-K phosphate buffer). A second buffer was
KR-Tes,1 which is a
Krebs-Ringer/Tes solution containing, in the following final millimolar
concentrations: NaCl 119; KCl 4.7; CaCl2 2.5; KH2PO4 1.2; MgSO4 1.2; and (sodium)
Tes, pH 7.4, 22.6. Crystalline bovine liver catalase was dissolved in
KR-Tes, concentrated on CF-25 ultrafiltration cones (Amicon), and then
washed on the cones (12) with the buffer to be used in each experiment.
All other enzymes were both dissolved and washed with the buffer to be
used in each experiment.
The solution of each enzyme was assayed for protein concentration (24)
and activity. The activity of catalase was determined from the
first-order rate constant of the rate of disappearance of
H2O2, at an initial concentration of 10 mM, as measured by absorbance at 240 nm with a recording
spectrophotometer (25). By this assay, the bovine liver catalase had an
activity of 23.7 s
1 per micromolar catalase concentration
(5.9 s
1 per micromolar heme concentration). The rate of
formation of H2O2 by glucose oxidase in the
presence of 5 mM glucose was determined with the assay for
H2O2 of Green and Hill (26). The glucose oxidase catalyzed the generation of H2O2 at an
average rate of 2,100 µmol min
1 per µmol of glucose
oxidase. The rate of generation of O
2 by xanthine oxidase in
the presence of xanthine (100 µM) was measured in 50 mM Na-K phosphate buffer, pH 7.4, by the rate of reduction of ferricytochrome c (10 µM) at 550 nm, using
the extinction coefficient at 550 nm of 21,000 M
1 cm
1, according to the method
of McCord and Fridovich (27). The replotting method of Sawada and
Yamazaki (28) provided assurance that the spontaneous dismutation
reaction was second-order with respect to O
2 and revealed a
value of 9.14 × 10
4 M s for
kd/kc2, in which
kd is the rate constant for the spontaneous dismutation reaction and kc is the rate constant for the reduction of cytochrome c by O
2. The factor
kd/kc2 provided a
means for correcting for the small fraction of O
2 that
undergoes spontaneous dismutation before the O
2 can reduce cytochrome c. The rates of generation of O
2, as
measured by the rate of cytochrome c reduction, were similar
whether catalase (2 µM) was present or absent. At pH 7.4 and 37 °C, the xanthine oxidase had an average specific activity of
115 µmol min
1 per µmol of enzyme. The total rate of
production of H2O2 by xanthine oxidase was
determined from the rate of production of urate, as measured by the
rate of increase in absorbance at 295 nm (29), and was confirmed with
the assay for H2O2 of Green and Hill (26) on
incubations containing xanthine oxidase, xanthine, and superoxide dismutase. The rate of oxidation of NADPH was determined from the
amount of 6-phosphogluconate formed, as described previously (12). All
reaction mixtures had a final volume of 1.0 ml. All incubations were at
pH 7.4 and 37 °C except for one experiment in which the conditions
were otherwise stated. The reaction components were at the final
concentrations indicated within parentheses when the concentrations
were not specified and were as follows: catalase and, when present,
xanthine oxidase, xanthine (100 µM), superoxide
dismutase, NADP+ (2 µM), glucose 6-phosphate
(1 mM), glucose-6-phosphate dehydrogenase (10 µg/ml),
glucose (5 mM).
For following the kinetics of reactions, readings of absorbance from
the spectrophotometer were taken on each group of cuvettes at intervals
of 1 min per group and were automatically stored in a computer for
later statistical analysis. For obtaining spectra of catalase,
absorbance readings were taken and stored at intervals of 1 nm. The
absorption spectra were obtained with a Beckman DU-7 spectrophotometer
at a recording speed of 1,200 nm min
1. Prof. Peter
Nicholls kindly provided the absorbances of equimolar concentrations of
ferricatalase (the resting or free form of catalase) and compounds I,
II, and III at intervals of 1 nm between 350 and 750 nm. The plots from
these absorbances were similar to previously published spectra (22,
30). The spectra for ferricatalase, compound I, and compound II were
confirmed in the following manner. One ml of 100 mM
potassium Ches buffer, pH 8.6, was added to 0.5 ml of the bovine liver
catalase from the bottle (20 mg/ml) to dissolve all enzyme crystals.
The dissolved enzyme was further diluted in 50 mM Na-K
phosphate buffer, pH 6.5, to a concentration of about 10 µM. One ml of enzyme preparation was then transferred to
a cuvette. After a preincubation period of 5 min at 37 °C, the
spectrum of ferricatalase was obtained. After the first spectrum was
obtained, 5 µl of 3% peracetic acid were added to the cuvette, and
the spectrum was immediately re-determined (compound I). One microliter
of 60 mM potassium ferrocyanide was then added to the same
reaction mixture, and after 10 min of incubation at 37 °C, the
spectrum was determined again (compound II). The absorbances of
equimolar concentrations of the four forms of catalase were used to
determine the amount of each form in H2O-treated catalase by
a least squares method, as follows. The minimum was found for the sum
(from 501 to 750 nm) of the squares of the difference between
Un and
ffAn + fIBn + fIICn + fIIIDn, in which n
is the wavelength, Un is the absorbance of the
treated catalase at wavelength n, and
ff through fIII represent
the unknown fractions of the treated catalase that are in the form of
ferricatalase, compound I, etc. An through
Dn represent the known absorbances of equimolar concentrations of ferricatalase through compound III at wavelength n. The value for each f was obtained from the
solution of the resulting simultaneous, linear equations for the
fs. A visual check of the validity of the result was
obtained with a spreadsheet/graphing program that allowed a comparison
of the observed spectrum with the spectrum from any specified
combination of the four forms of catalase. The formation of compound II
is usually followed at 435 nm, the isosbestic point between
ferricatalase and compound I (22). The difference in extinction
coefficient between ferricatalase and compound II was considered to be
32 mM
1 cm
1 (31). As observed
also by Chance (30), the extinction coefficient of compound III at 435 nm was found to be similar to that of compound II. The increases in
absorbance at 435 nm were therefore considered to represent the
combined rate of formation of both compounds.
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RESULTS |
Steady-state Concentrations of H2O2 and
O
2--
The experimental conditions were set as follows so as
to be biologically realistic: the pH was 7.4; the temperature was
37 °C; and the catalase concentration (2-3 µM) was
similar to that of human erythrocytes (11). The rates of
H2O2 and O
2 generation were 2-15 nmol
ml
1 min
1, which are similar to the
estimated rate of H2O2 generation in the human
erythrocyte under resting conditions and under peroxidative stress,
respectively (9). Under these conditions, the concentrations of
H2O2 and O
2 were too low to be
determined by present methods, but they could be determined indirectly.
Whether generated by glucose oxidase or xanthine oxidase,
H2O2 rises to a steady-state concentration at
which the rate of removal in the presence of catalase (Reaction 1)
equals the rate of generation.
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(Reaction 1)
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The bovine liver catalase was found to have a specific activity
(see "Experimental Procedures") that would result in a steady-state nanomolar concentration of H2O2 of 0.7 Vh/C, in which
Vh is the total rate of generation of
H2O2 in nmol ml
1
min
1 and C is the micromolar concentration of
the catalase.
When O
2 was generated by incubation of 40 nM
xanthine oxidase with xanthine (100 µM), the xanthine
oxidase produced uric acid at an average rate of 8.8 nmol
ml
1 min
1 but O
2 at an average rate
of only 4.6 nmol ml
1 min
1. This result
indicated that 74% of the oxidation of xanthine by xanthine oxidase
under these conditions was leading to the direct production (29) of
H2O2 (Reactions 2 and 3), whereas the remainder
was resulting in the production of O
2 (Reaction 4).
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(Reaction 2)
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(Reaction 3)
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(Reaction 4)
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Results of the cytochrome c assay indicated that 1 µM manganese superoxide dismutase reduced the net rate of
generation of O
2 from 40 nM xanthine oxidase
(Reaction 5) by 97%.
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(Reaction 5)
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O
2 was assumed to reach a steady-state concentration at
which the rate of removal of O
2, whether by superoxide
dismutase or spontaneous dismutation (Reaction 5), equaled the rate of
generation of O
2. The second-order spontaneous dismutation
rate of 4.8 × 105 M
1
s
1 at room temperature (31) was calculated to be 6.0 × 105 M
1 s
1 after
adjustment to 37 °C (32), indicating that a rate of O
2 production of 4.6 nmol ml
1 min
1 should
result in a steady-state concentration of 360 nM for
O
2 in the absence of superoxide dismutase. As expected from
Reactions 2-R5, the production of H2O2 by
xanthine oxidase in the presence of manganese superoxide dismutase
reached a steady-state rate similar to the rate of production of uric
acid. For 40 nM xanthine oxidase and 2 µM
catalase, the concentration of H2O2 would have been 3 nM.
Determinations of Compounds I, II, and III--
Changes in the
absorbance at 435 nm are traditionally used to follow the kinetics of
compound II formation and removal. Changes at this wavelength, however,
also reflect changes in the concentration of compound III. Computerized
analysis of the absorption spectrum of bovine liver catalase at various
times in the incubation gave the percentage of catalase that was in
each of the four states of the enzyme (see "Experimental
Procedures") and thereby revealed the extent to which absorbance
changes at 435 nm were essentially measures of compound II alone. At a
xanthine concentration of 100 µM, the generation of
O
2 and H2O2 by 40 nM
xanthine oxidase ended between 10 and 20 min after the start of the
reaction, when the xanthine was depleted (Fig.
2). During this exposure, the percentage
of catalase in the native state (ferricatalase) fell to a minimum of
49%. The percentage as compound III rose initially more rapidly than
did compound II but reached a maximum of only 9% (Fig. 2). In
contrast, compound II rose steadily to a maximum of 33% at 20-24 min.
The increase in absorbance at 435 nm provided an estimate of the
combined percentage of compound II and compound III (see
"Experimental Procedures"), and this estimate was in general
agreement with the combined percentage as determined by the
computerized analysis of the absorption spectrum from 501 to 750 nm
(Fig. 2). This agreement served to confirm that the difference in molar
(heme) specific absorbance between ferricatalase (or compound I) and
compound II at 435 nm was 32,000, that compound III had a similar molar
specific absorbance at 435 nm, and that the bovine liver catalase used
in this study had four functional heme groups. This information, in
turn, allowed comparison of the loss in activity of bovine liver
catalase and the formation of compounds II by both
H2O2 and O
2. In results not given,
this comparison confirmed that compound II has no catalase activity at
pH 7.4 and at 37 °C, as had been demonstrated earlier under other
conditions by Chance (33).

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Fig. 2.
Percentages of four forms of catalase during
exposure to O 2 generated by xanthine oxidase. The
reaction was at 37 °C with bovine liver catalase (3 µM) and Na-K phosphate buffer (50 mM), pH
7.4. The results are averages of incubations at 3 different times. The
presence of xanthine (100 µM) and xanthine oxidase (40 nM) caused the generation of O 2 at a rate of 4.2 nmol ml 1 min 1 and
H2O2 at a total rate of 8.0 nmol
ml 1 min 1. Percentages of the four forms of
catalase were determined by a least squares method (see "Experimental
Procedures") from absorbances from 501 to 750 nm. ×, the
combined percentage of compound II and compound III, as determined by
changes in absorbance at 435 nm.
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The percentage of catalase in each of its four forms was determined at
intervals for a variety of reactions. Table
I gives the results only at either 8 or
60 min of reaction time. The ratio of compound I to ferricatalase was
lower when the catalase was exposed to the action of xanthine oxidase
(Table I, row e) than when it was exposed to the action of glucose
oxidase (Table I, rows a and c). The percentage as compound II was
lower in the four reactions containing NADPH than in the corresponding
reactions without added NADPH. At 8 min, less compound III was present
in those reactions in which only H2O2 was
generated (Table I, rows a, b, f, and h) than in those
reactions in which O
2 was also generated (Table I, rows e, g,
and i). The presence of ethanol (2 mM) essentially
eliminated the presence of compounds I and II but allowed the
generation of some compound III (Table I, row i).
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Table I
Percentages of the four forms of bovine liver catalase after exposure
to H2O2 generated by glucose oxidase or to O 2
and H2O2 generated by xanthine oxidase
Catalase, at a final concentration of 3 µM, was exposed
at pH 7.4 and 37 °C to glucose oxidase (Gox), 6.7 nM,
generating H2O2 at a rate of 14.8 nmol ml 1
min 1, or to xanthine oxidase (Xox), 40 nM,
generating H2O2 at a rate of 6.8 to 8.9 nmol
ml 1 min 1, and O 2 at an average rate of 4.1 nmol ml 1 min 1. Other final concentrations were as
follows: manganese superoxide dismutase (SOD), 1 µM;
NADPH, 2 µM; and ethanol, 2 mM. NADPH was
kept in the reduced state by the presence of glucose-6-phosphate
dehydrogenase and glucose 6-phosphate (see "Experimental
Procedures"). The reaction time was 8 min except for that of rows c
and d which was 60 min.
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Kinetics of the Formation and Disappearance of Compound
II--
Results of the previous section indicated that changes in the
absorbance of catalase at 435 nm were essentially those from compound
II except during the 1st min after the generation of O
2, when
a small amount of compound III was formed. The formation of compound II
was followed by recordings of the absorbance at 435 nm of 2 µM catalase during incubation under the various
conditions described in the legend of Fig.
3. Drops in absorbance at the start of
the reaction at 5 min were caused by the slight dilution resulting from
the addition of the starting solution. The absorbance increases at 435 nm, such as those of Fig. 3, were found by computerized and visual
curve fitting to follow Equation 1
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(Eq. 1)
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for a reaction in which the formation of compound II, at a
constant rate of VII, is offset by the
first-order decay of compound II, with a decay constant of
k. The visual curve-fitting method was used to determined
provisional values for VII and k,
then the final values were determined from iteration for a least
squares fitting of the increases in absorbances at 435 nm between 1 and 9 min after the start of the reaction. These calculations indicated that NADPH reduced the rate of compound II formation
(VII) from H2O2 by an
average of 82%, when the H2O2 was generated by
glucose oxidase in three experiments of the type shown in Fig.
3A, and by an average of 83% when the
H2O2 was generated in five experiments by
xanthine oxidase in the presence of superoxide dismutase, as in Fig. 3,
B and C. The difference between the two curves
for the xanthine oxidase reaction, with and without superoxide
dismutase (Fig. 3B), was considered to represent the
contribution to the rate of formation of compound II that was
attributable only to O
2. The difference between the two curves
in Fig. 3B was similar to the difference between the two
curves in Fig. 3C, indicating that NADPH had very little
effect on the rate of generation of compound II that could be
attributed to O
2 alone. In five experiments of the type shown
in Fig. 3, B and C, NADPH decreased the
difference between the VIIs (with and without superoxide dismutase) by
an average of 11 ± 12% (±S.E. of the mean). Results similar to
those of Fig. 3, B and C, were obtained with 40 nM xanthine oxidase and Cu,Zn superoxide dismutase and, at
a 5-fold slower rate of O
2 generation, with 8 nM
xanthine oxidase and manganese superoxide dismutase (results not
given). In contrast to the large effect of NADPH in lowering the rate
of compound II formation by H2O2, NADPH only
mildly affected the rate of decay of compound II (Table II).

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Fig. 3.
Changes in absorbance of catalase at 435 nm
during exposure to O 2 and H2O2.
The conditions were those of rows a-h of Table I except that the
catalase concentration was 2 µM. A, , blank
in which glucose was omitted. For purposes of graphic clarity, the
blanks of B and C, which resembled that of
A, were omitted, and the absorbances of the lower
two curves of A and the lower curve of
B and C were decreased by 0.01. SOD,
superoxide dismutase.
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Table II
Rate constants for disappearance of compound II
Compound II was generated at 37 °C and pH 7.4 by the addition of
xanthine to a reaction mixture containing xanthine oxidase and bovine
liver catalase. Concentrations immediately after the addition were
xanthine, 100 µM, xanthine oxidase, 4 nM, and
catalase, 2 µM. Measurements of urate production
indicated that the xanthine had been consumed by 15 min of reaction
time. Additions, to the final concentrations indicated below, were made
at 15 min of reaction time. The first-order rate constant,
k, for compound II disappearance was calculated from the
absorbance at 435 nm between 28 and 38 min after the start of the
reaction. The means (±S.D.) are from five replicates.
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Stoichiometry of NADPH Oxidation and Compound II
Formation--
NADP (2 µM) was kept in the reduced form
(NADPH) at a steady-state concentration of 2 µM in the
present study by the addition of a relative surplus of glucose
6-phosphate and glucose-6-phosphate dehydrogenase. When NADPH is
generated in this way, the amount of 6-phosphogluconate present at the
end of an incubation serves as a measure of how much NADPH was
oxidized. In Fig. 4, the amount of NADPH
oxidized during the first 10 min of the reaction is compared with the
amount of compound II and compound III formed over the same interval.
The amount of compounds II and III formed, and the amount of NADPH
oxidized, was only a small fraction of the amount of
H2O2 and O
2 generated over the 10-min
period in the various experiments of Fig. 4 (approximately 72 and 40 nmol ml
1, respectively). Conversely, the amount of NADPH
oxidized was greater than the amount of compounds II and III that was
formed in the absence of NADPH and was decidedly greater than the
amount by which the presence of NADPH decreased the formation of
compounds II and III (Fig. 4). In contrast, the amount of NADPH
oxidized was less than the amount by which compounds II and III decayed in the absence of H2O2 and O
2 (but
more than the amount by which NADPH increased the decay of compounds II
and III) (Fig. 4). As with the rates of formation of compound II in
Fig. 3, B and C, the differences in micromolar
concentrations of compounds II and III at 10 min, caused by superoxide
dismutase (Fig. 4), had a value (1.02 µM), when NADPH was
absent, that was similar to the corresponding difference (1.07 µM) when NADPH was present. Superoxide dismutase,
however, reduced the amount of NADPH oxidized in 10 min (Fig. 4). The
reduction was from 4.05 to 3.17 nmol ml
1, which was
significant at the p = 0.01 level. The results of row i
of Table I indicated that essentially only ferricatalase and compound
III were present when ethanol was added to the reaction mixture. The
ability of NADPH to modify the direct formation of compound III from
ferricatalase by O
2 (step 4 of Fig. 1) was therefore evaluated by observing the effect on catalase of the xanthine
oxidase reaction in the presence of ethanol (Fig. 4). Before conversion
to nmol ml
1 in 10 min, the increase in absorbance at 435 nm was corrected for the drop in absorbance resulting from the addition
of the starting solution of xanthine, as observed in a control solution to which an equivalent volume was added as water. Although low, the
concentration of compound III was less when NADPH was present. Without
added NADPH, the concentration of compound III reached a (heme)
concentration 0.103 ± 0.027 µM (mean ± S.D.)
at 10 min, corresponding to 1.3% of the catalase. The concentration of
compound III at 10 min with added NADPH was 0.027 ± 0.021 µM. The difference was significant (4 degrees of freedom,
t = 3.9) at the level of 0.01 < p < 0.025. A second method for determining the stoichiometry between
NADPH oxidation and compound II formation is that of Hillar and
Nicholls (16) of adding different amounts of NADPH, measuring the
duration of the lag before the generation of compound II begins, as
measured by absorbance at 435 nm, and determining the slope that
follows the lag. Because of differences in interpretation (see
"Discussion"), an experiment of this type by Hillar and Nicholls (16) was repeated (Fig. 5). The concavity
at the beginning of the upsweep of curves b, c, and
d was due to the presence of NADP+. Although
NADP+ is a weak competitor of NADPH (12), the ratio of
NADP+ to NADPH is very large just before the NADPH is
exhausted. The duration of the lag of reactions b, c, and d of Fig. 5
indicated that NADPH was being oxidized at an average rate of 0.288 nmol ml
1 min
1. The average rate of compound
II formation in reactions b, c, and d was 0.073 nmol ml
1
min
1, giving a ratio of NADPH oxidation to compound II
formation of 3.9. The same experiment at pH 7.4 gave a ratio of 5.1 (figure not shown).

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Fig. 4.
The amounts of compounds
(Cpds) II and III formed, and NADPH oxidized, over 10 min in reactions in which H2O2, or O 2,
and H2O2 were generated. Glucose oxidase
(Gox) was present at a final concentration of 4.1 nM and generated H2O2 at a rate of
7.2 nmol ml 1 min 1. Xanthine oxidase
(Xox) was present at a final concentration of 40 nM and generated H2O2 and
O 2 at rates of 7.9 and 4.0 nmol ml 1
min 1, respectively. Other final concentrations were
catalase, 2 µM; manganese superoxide dismutase
(SOD), 1 µM; ethanol, 2 mM; and
NADPH 2 µM. The amount of NADPH oxidized was determined
from the amount of 6-phosphogluconate generated. The estimates for the
amount of NADPH oxidized were corrected for the formation of NADPH from
the NADP+ (2 µM) initially present and for
the amount of 6-phosphogluconate generated in 10 min in blank reactions
(averaging 0.8 nmol ml 1 in the blank consisting of
catalase without glucose oxidase and 1.2 nmol ml 1 in the
blank consisting of xanthine and xanthine oxidase without catalase).
The number above each column is the standard deviation based
on the number of replicates shown in parentheses.
|
|

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Fig. 5.
Lag in compound II formation caused by added
NADPH. The reaction mixtures were incubated at 25 °C and
contained, in the following final concentrations: potassium phosphate
buffer, pH 6.5, 10 mM; beef liver catalase, 1.0 µM; glucose oxidase, 2 nM; glucose, 4 mM; and NADPH at (curve a) 0.0, (curve
b) 3.3, (curve c) 6.6, and (curve d) 10.0 µM. Arrow, start of the reactions by the
addition of glucose.
|
|
 |
DISCUSSION |
Previously Proposed Model of the Action of NADPH--
Soon after
the discovery that each subunit of bovine catalase has one tightly
bound molecule of NADPH (11), the x-ray crystallographic data on the
three-dimensional structure of bovine liver catalase was re-evaluated,
and the NADPH was found to be on the surface of the enzyme,
approximately 20 Å from the heme group (15). Because NADPH is too
large to reach the latter through the relatively narrow channel leading
to the heme group, electron tunneling was assumed to be the mechanism
by which NADPH is able to reduce compound II to ferricatalase (18).
Olson and Bruice (19) used computerized calculations, along with
information from the known three-dimensional structure of bovine liver
catalase, to estimate the probable route of the electron tunneling.
Others have also obtained information on the probable route of electron
tunneling between the bound NADPH and the heme center of bovine or
Proteus mirabilis catalase (18, 34, 35). Using time-resolved
x-ray crystallography and single crystal microspectrometry, Gouet
et al. (20) determined the structure of compound I and
compound II of NADPH-dependent catalase from Proteus
mirabilis, including the formation and transformation of the
ferryl groups. An unexpected result of their study was the finding that
compound I acquired an anion at a site near neither the heme iron nor
the NADPH-binding site. The most likely candidate for the anion seemed
to be O
2 (20). Hillar et al. (16, 17) proposed that
NADPH reduces neither compound I nor compound II but rather a
postulated intermediate between the two (Fig.
6). The findings of Ivancich et
al. (36), who used EPR and rapid mix freeze-quench techniques,
provided some support for the presence of such an intermediate. NADPH
functions in its traditional role as a two-electron reducing substance
in step 10 of the model of Hillar et al. (17)
(Fig. 6), whereas Almarsson et al. (18) propose that NADPH
leads to one-electron reduction of compound II via electron tunneling,
the other electron going elsewhere, such as to O2 to form
O
2.

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Fig. 6.
Sequence for prevention of compound II
formation by NADPH. This scheme replaces the reactions shown on
the right in Fig. 1. Except for step 3, the
representations and reactions are those of Hillar et al.
(17). AH represents a postulated oxidizable amino acid near
the heme group (17). An intermediate, formed by step 3a, is
reduced by NADPH via electron tunneling.
|
|
Support for the Intermediate State--
The present authors (12)
have said that NADPH both decreases the formation of compound II and
increases the rate of removal of compound II. In contrast, Hillar
et al. (16, 17) state that NADPH decreases the rate of
formation compound II but does not increase its rate of removal. Both
actions of NADPH were observed in the present study, but decreasing the
rate of formation of compound II was the predominant action. NADPH only
mildly increased the rate of spontaneous decay of compound II (Table
II). The decay constant of compound II in the presence of NADPH was 1.4 times the constant without NADPH (Table II). The least squares fitting of the absorbances of Fig. 3A (upper curve) to
Equation 1 provided an estimate of the decay constant and initial rate
of increase in concentration of compound II. Equation 1 revealed that
the concentration of compound II at 10 min of reaction time would have
been reduced by only 12% by increasing the decay constant by a factor
of 1.4. In contrast, NADPH actually decreased the concentration by 80%
(Fig. 3A). Moreover, reconsideration of our results at pH
6.5 (12) in the same manner indicates that the action of NADPH at that
pH was also one of preventing the formation of compound II by
H2O2, rather than increasing the rate of
removal of compound II. That NADPH prevents the formation of compound II by H2O2, rather than increases the decay of
compound II, is supported also by the unexpected finding, in the
present study, that NADPH has a limited ability to offset the formation
of compound II that arises from the primary action of O
2. Had
the protective action of NADPH been due largely to increasing the rate
of removal of compound II, then NADPH should have provided protection
against both H2O2 and O
2. The results
in Table I show that NADPH, unlike ethanol, does not prevent the
formation of compound II by lowering the concentration of compound I. By documenting that NADPH prevents the formation of compound II, rather
than increases the rate of removal of compound II, the present study
strengthens the claim of an intermediate state of bovine liver
catalase, between compound I and compound II. It is difficult to
explain the preventive role of NADPH without assuming the presence of
such an intermediate.
Modification and Expansion of the Model--
If the mechanism of
an intermediate, as proposed by Hillar et al. (17), is
accepted, then the findings of the present study would require that
O
2 reacts directly with compound I to produce compound II,
by-passing the intermediate in the process (Fig. 6). Hillar et
al. (16, 17) found that ferrocyanide increased the rate of
formation of compound II through what they assumed to be the
one-electron reduction of the intermediate by ferrocyanide at
step 3b (see Fig. 6). Since they found that NADPH greatly
reduced the rate of compound II formation by ferrocyanide, it is
unlikely that the action of O
2 is at step 3b. In
their two articles on the concept of an intermediate, and in support of
that concept, Hillar et al. (16, 17) claimed that the rate
of oxidation of NADPH was similar to the rate at which compound II was
formed in the absence of NADPH. When corrected for the molar specific absorbances of NADPH and compound II, however, the rate of NADPH oxidation can be shown to be approximately 3 times the rate of compound
II formation in their first article (16) and even higher in their
second report (17). Fig. 5, in fact, is a repeat of the experiment of
Hillar and Nicholls (16) and reveals a ratio of 3.9. When the rate of
NADPH oxidation is measured by the rate at which 6-phosphogluconate is
formed by the NADPH-generating system, the ratio of NADPH oxidation to
compound II formation is 3 to 4 at pH 7.4 (Fig. 4) and pH 6.5 (12) and
at different rates of H2O2 generation (12).
Thus, a ratio of 3 or more has been demonstrated at two pH values, by
two methods, and at different rates of H2O2
production. As indicated in the legend of Fig. 4, very little NADPH was
oxidized when H2O2 and O
2 were
generated in the absence of catalase. For detection of an effect of
substances of low molecular weight, such as ions of trace metals that
might accompany the catalase, these control incubations contained an ultrafiltrate of the catalase. The volume of the ultrafiltrate was the
same as the volume of catalase solution added to the incubation mixtures. Moreover, these control incubations provide an overestimate of the ability of H2O2 to oxidize NADPH in the
absence of catalase, since the H2O2 would reach
concentrations well above those in the presence of catalase. Very
little oxidation of NADPH occurred when catalase was exposed to
H2O2 and O
2 in the presence of ethanol (Fig. 4) and therefore at extremely low concentrations of compound I
(Table I). Reversibility of step 3a of Fig. 6 could cause
the rate of NADPH oxidation to exceed the rate at which compound II would be formed in the absence of NADPH. Specifically, the
reversibility would need to result in a relatively rapid
interconversion of compound I and the intermediate. Computer
simulations of the schemes of Figs. 1 and 6, however, indicate that
this reversibility would cause the curve for compound II formation to
have two distinct slopes and therefore to fail to fit Equation 1.
Although the steady-state concentration of compound III is low, the
concentration is even lower in the presence of NADPH, even under
conditions when essentially no compound I or compound II is present
(Table I and Fig. 4).
Almarsson et al. (18) point out that compound I is an
oxidant and also an unstable species, tending to engage in the side reaction of becoming compound II when the encounter between compound I
and H2O2 is delayed. Such a delay occurs under
physiological conditions, when the rate of generation of
H2O2 is low. That compound I is a strong
oxidant is underscored by the fact that compound I oxidizes
H2O2 in the normal cycle of
H2O2 disposal (Fig. 1). Compound I leads to the
oxidation of a reductant within the structure of catalase, the
so-called endogenous donor, causing compound I to become compound II.
We wish to suggest that NADPH may protect catalase from oxidative
damage through actions broader than merely preventing the formation of
compound II. NADPH may lead to the reduction of oxidizing states and
internal groups of catalase other than the intermediate, possibly
including a small percentage of compound I, itself. This broader action
of NADPH could account for the oxidation of NADPH at a rate exceeding
by severalfold the rate at which compound II would otherwise be formed.
 |
ACKNOWLEDGEMENT |
We thank Prof. Peter Nicholls for providing
the absorption spectra of equimolar concentrations of the four forms of
bovine liver catalase as well as for suggestions on converting the
enzyme to compounds I, II, and III.
 |
FOOTNOTES |
*
This work was supported by funds from P. F. Biotecnologie (CNR Target Project on Biotechnology), MURST 1996-1997,
and Ministero Sanità RF 96.286/ICS070.1.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Department of
Pediatrics, University of Noth Carolina, Chapel Hill, NC 27599-7487. Tel.: 919-966-1447; Fax: 919-966-9042.
 |
ABBREVIATIONS |
The abbreviations used are:
Tes, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid;
Ches, cyclohexylaminoethanesulfonic acid.
 |
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