(Received for publication, December 29, 1994; and in revised form, January 26, 1995)
From the
Phagocytic cytochrome b is a unique
heme-containing enzyme, which catalyzes one electron reduction of
molecular oxygen to produce a superoxide anion with a six-coordinated
heme iron. To clarify the mechanism of the superoxide production, we
have analyzed oxidation-reduction kinetics of cytochrome b
purified from porcine neutrophils by
stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b
was rapidly reoxidized by O
showing spectral changes with clear isosbestic points, which were
also observed during the reduction of ferric cytochrome b
with Na
S
O
under anaerobic conditions. The single turnover rate for the
reaction with O
linearly depended on the O
concentration but was not affected by addition of CO. The rate of
the reaction decreased with an increase of pH giving a
pK
of 9.7. Under complete anaerobic
conditions, ferrous cytochrome b
was oxidized by
ferricyanide at a rate faster than by O
. The thermodynamic
analysis shows that the enthalpic energy barriers for the reactions of
cytochrome b
are significantly lower when
compared to the autoxidation of native and modified myoglobins through
the formation of the iron-O
complex. These findings are
most consistent with the electron transfer from the heme to O
by an outer-sphere mechanism.
Molecular oxygen is crucial for oxidative phosphorylation and
other important chemical reactions in living cells. In many processes
of the metabolism, O is transformed to reduced or activated
forms by heme-containing enzymes. Phagocytic cells such as neutrophils
and macrophages also use O
to produce activated oxygen
species, including hypochlorous acid and hydroxyl radicals, for killing
microorganisms during phagocytosis. The consumption of O
,
called ``respiratory burst,'' is accompanied by oxidation of
NADPH and production of superoxide anions
(O
) (1, 2, 3, 4, 5) . The
O
-producing reaction is catalyzed by a
membrane-bound electron-transferring enzyme system, ``NADPH
oxidase,'' in which cytochrome b
is the
only identified component that contains redox
centers(6, 7, 8, 9, 10, 11, 12) .
In previous studies(13, 14) , we have demonstrated
catalytic production of O
by cytochrome b
purified from porcine neutrophils in an
artificial reconstitution system with an exogenous reductase. Purified
cytochrome b
had a midpoint reduction potential (E
) (
)of -255 mV at pH 7.4 and
showed low spin EPR signals at 10 K. In stopped-flow measurements, the
reduced form of cytochrome b
was reoxidized to
the ferric form by O
according to a second-order reaction
with a rate constant (k
) of
10
M
s
at 10 °C.
From these results, we have concluded that the heme in a
six-coordinated low spin state catalyses one electron reduction of
O
.
All known heme-containing enzymes involved in
reduction or activation of O, such as cytochrome c oxidase or cytochromes P-450, have a heme in which the five
coordination sites of iron are occupied by intrinsic ligands, and the
sixth coordination site is opened for binding O
or other
extrinsic ligands. The heme of these enzymes forms an iron-O
complex during the reduction of O
, or forms the
complexes with respiratory inhibitors. On the other hand, the
respiratory burst has been known to be insensitive to the respiratory
inhibitors, and cytochrome b
in situ does not form a complex with CO(15) . Judging from
spectroscopic data, the heme of cytochrome b
is
in a low spin six-coordinate state in both the ferric and ferrous
forms(12, 14, 15, 16, 17) .
Moreover, we confirmed that the respiratory inhibitors gave no effect
on the absorption spectra nor on the catalytic activity of purified
cytochrome b
in the reconstituted
system(14) . These facts indicate strong coordination of both
the axial ligands to the heme iron and suggest that cytochrome b
reduces O
by a unique mechanism
distinct from those of other heme-containing enzymes.
The purpose of
this study is to examine whether an oxygenated intermediate is formed
during the reduction of O by ferrous cytochrome b
or not and to clarify the mechanism of the
reaction. The reduced cytochrome was mixed with an oxygenated solution
under various conditions, and the spectral changes during the reaction
were measured on the millisecond time scale by rapid-scanning
spectroscopy. The temperature and pH dependence of the reaction was
determined. Under anaerobic conditions, the ferrous cytochrome showed
the ability to react with artificial oxidants. The properties of the
reactions by cytochrome b
are compared with
those by other heme proteins. The results obtained here, together with
our previous ones(13, 14, 15) , suggest that
the reduction of O
by cytochrome b
occurs via a peripheral process, i.e. electrons being
transferred from the heme to O
at or near the heme edge
without formation of the iron-O
complex.
Superoxide-producing cytochrome b was
purified from porcine neutrophils according to the method described
previously(14) , except that the gel filtration chromatography
was performed with Superdex 200 pg (Pharmacia) packed into a 1.6
60-cm column. The final purification step with heparin affinity
chromatography was omitted. The purified preparation was depleted of
flavins (FAD/heme <0.01) as described previously(14) . The
concentration of cytochrome b
was determined
using a reduced-minus-oxidized extinction coefficient of 21,600 M
cm
at 558
nm(18) .
Tetrazole myoglobin (Tet-Mb) was prepared and purified according to Shiro et al.(19, 20) . The time course of autoxidation of Tet-Mb was measured by following the absorbance change at 570 nm as described previously(19) .
Measurements of spectral changes of cytochrome b in a time range of milliseconds were carried out with a
stopped-flow and rapid-scanning spectrophotometer, Unisoku type
RSP-601. The purified cytochrome was diluted with a buffer solution
containing 100 mM HEPES-NaOH, pH 7.4, and 0.1% sucrose
monolaurate, unless otherwise noted in the text. The sample solution
was depleted of O
by gently bubbling N
gas in
one of the two reservoirs of the stopped-flow mixer. In the other
reservoir, the buffer solution containing reagents mentioned in the
text was bubbled with a gas mixture of N
and O
,
which was prepared with a Estec SGD-SC standard gas divider, for at
least 15 min. In measurements of the reactions with artificial
oxidants, both the solutions also contained 100 mM glucose, 10
units/ml glucose oxidase (Sigma), 2 units/ml catalase (Sigma), and 50
µg/ml superoxide dismutase (Sigma) and were bubbled with N
gas to completely remove O
. The rapid-scanning
recording of a spectrum took 1 ms/208 nm, and 512 spectra were measured
with or without time intervals after mixing of the solutions. The dead
time was estimated at about 6 ms based on the time course of reduction
of dichlorophenolindophenol by ascorbate in separate measurements under
the experimental conditions used. Concentration of O
in the
reaction mixture was estimated by amounts of oxygenated
myoglobin(Fe
) formed after mixing deoxy
myoglobin(Fe
) with an oxygenated buffer in other
separate measurements. Temperature was controlled by circulating water
maintained at constant temperature with a Lauda thermostat type RM6
around the reserved solutions and the flow lines inside the mixer.
The time course data for the oxidation of ferrous cytochrome b by oxidants were analyzed according to the
equation based on second-order reactions as described
previously(14) . The change in concentration of cytochrome b
from the ferrous form to the ferric form was
estimated based on the reduced-minus-oxidized extinction coefficient at
426 nm of 110,000 M
cm
.
The ferric form of cytochrome b was
rapidly reduced to the ferrous form with absorption maxima at 558, 528,
and 425 nm after mixing with an excess amount of
Na
S
O
under anaerobic conditions (Fig. 1). The spectral changes clearly showed isosbestic points
at 417.4 and 438.2 nm. The time course data were fitted with a single
exponential curve based on a pseudo-first-order reaction (not shown),
and the apparent rate constant was estimated at 3.6 s
under the experimental conditions (see the legend of Fig. 1).
Figure 1:
Spectral changes during the
reduction of cytochrome b with
Na
S
O
under anaerobic conditions.
Cytochrome b
was mixed with a
Na
S
O
solution at the final
concentrations of 2.8 µM and
1 mg/ml, respectively,
in a stopped flow apparatus, and the spectral changes were measured by
a rapid-scanning spectrophotometer at 10 °C. The spectra recorded
at 0, 60, 120, 180, 240, 300, 360, and 420 ms after the mixing are
shown. Inset, the spectral changes in the Soret band are
displayed as the difference spectra against that at 0 ms after the
mixing.
The ferrous form of cytochrome b prepared by addition of a slight excess of
Na
S
O
was mixed with an oxygenated
solution prepared at a low concentration of O
, and the
spectral changes during the reaction of cytochrome b
with O
were measured on the millisecond time scale (Fig. 2). The initial spectrum of the ferrous form was gradually
replaced with that of the ferric form in the time domain. Clear
isosbestic points were observed at 417.6 and 437.6 nm, which were
identical with those in the spectral changes during the reduction with
Na
S
O
under the anaerobic conditions
within the wavelength resolution of the spectrophotometer (see Fig. 1).
Figure 2:
Spectral changes during the oxidation of
ferrous cytochrome b with O
.
Cytochrome b
reduced with a small excess of
Na
S
O
was mixed with an oxygenated
solution at the final cytochrome and O
concentrations of
3.5 and 10 µM, respectively, and the spectral changes were
measured at 2 °C. The spectra recorded at 0, 4, 8, 12, 16, 20, 40,
and 80 ms after the mixing are shown. Inset, the spectral
changes in the Soret band are displayed as the difference spectra
against that at 80 ms after the mixing.
The effect of O concentration on the
time course for the reaction was examined as shown in Fig. 3, a
d. The apparent rate increased with the O
concentration in the tested range. The addition of CO did not
affect the reaction (Fig. 3, e). The second-order rate
constants (k
) were almost constant under these
conditions and were estimated at 8.2
9.5
10
M
s
(see the
legend to Fig. 3). Effects of the addition of
CN
, N
, and pyridine
were not observed (not shown).
Figure 3:
Effects of O concentration and
addition of CO on the time course for the reaction of ferrous
cytochrome b
with O
. The reactions
were measured at 10 °C. Theoretical curves based on the
second-order reaction were fitted to the data (dots) with the
rate constants (k
) and the 0 ms concentrations of
the ferrous cytochrome b as follows: a, 9.3
10
M
s
and
1.13 µM; b, 9.5
10
M
s
and 0.96
µM; c, 8.5
10
M
s
and 0.69
µM; d, 8.2
10
M
s
and 0.59
µM; e, 9.5
10
M
s
and 1.05
µM, respectively. Addition of CO showed no inhibitory
effect on the reaction (e). The absorbance at time 0 ms
decreased with the concentration of O
by the oxidation
during the dead time of the stopped-flow measurements (see
``Experimental Procedures'').
The effect of pH on the reaction of
ferrous cytochrome b with O
was
examined between pH 7 and 11. In this pH range, little change in
absorption spectra for the ferric and ferrous forms was observed with
the variation of pH. The rate constant of the reaction was almost
constant over a pH range from 7 to 9, and decreased above pH 9 (Fig. 4). The pH dependence gives a single pK
of 9.7.
Figure 4:
Dependence of the rate constant on pH for
the reaction of ferrous cytochrome b with
O
. The reactions were measured at 10 °C in buffer
solutions which contained 0.1% sucrose monolaurate and 100 mM tris(hydroxymethyl)aminomethane-NaOH at pH between 7.8 and 9.0,
100 mMN-cyclohexyl-2-hydroxy-3-aminopropanesulfonic
acid-NaOH at pH 9.4 and 9.9, or 100 mMN-cyclohexyl-3-aminopropanesulfonic acid-NaOH at pH 10.5
and 11. Initial concentrations of O
in the reaction
mixtures were 10 µM at pH between 7.4 and 9.4, and 20
µM at pH between 9.9 and 11. Each data point is the
average of 4
8 separate measurements, and error bars indicate the
standard deviation from the average. The solid line was drawn
assuming that k
decreased from 8.8
10
to 2.4
10
M
s
with pK
of
9.7.
The ability of ferrous cytochrome b to react with artificial oxidizing agents was
examined under completely anaerobic conditions (Fig. 5). We
found that cytochrome b
was highly reactive with
ferricyanide (E
= +420 mV). The
spectral changes during the reaction (Fig. 5A) show
clear isosbestic points, which are also observed in the reaction with
O
. k
for the reaction with
ferricyanide was estimated at 6.4 ± 0.4
10
M
s
(4
measurements) at 10 °C. In addition, p-benzoquinone (E
= +285 mV), a rather hydrophobic
agent, was able to oxidize cytochrome b
. The
reaction, however, was much slower than that with ferricyanide (Fig. 5B), and k
was estimated at
3.5 ± 0.3
10
M
s
(three measurements) at 10 °C.
Figure 5:
Oxidation of ferrous cytochrome b by artificial oxidizing agents. A,
spectral changes during the oxidation of ferrous cytochrome b
with ferricyanide. Ferrous cytochrome b
was mixed with an anaerobic solution
containing ferricyanide at both final concentrations of 5
µM, and the spectral changes were measured at 5 °C.
The spectra recorded at 0, 4, 8, 12, 16, 20, 40, and 100 ms after the
mixing are displayed as the difference spectra against that at 100 ms
after the mixing. B, time courses for the oxidation of
cytochrome b
by p-benzoquinone (trace b) and by ferricyanide (trace c) at 10 °C.
Initial concentrations of ferrous cytochrome b
, p-benzoquinone, and ferricyanide were 5 µM, 5
µM, and 5 mM, respectively. No absorbance changes
were observed in the absence of oxidants (trace a), showing
that the reaction mixture was completely depleted of O
under the experimental conditions. k
was
estimated by fitting theoretical curves to these data (see
text).
The
temperature dependence of the reactions of ferrous cytochrome b with O
and ferricyanide was
determined at pH 7.4 (Fig. 6). For comparison, the dependence of
autoxidation of deoxy Tet-Mb was also determined under similar
experimental conditions. The thermodynamic parameters of activation for
these reactions were obtained on the basis of the transition state
theory (21) and listed in Table 1with those for the
reduction of ferric cytochrome c by
O
, which were calculated based on the
data of Butler et al.(22) (see
``Discussion'').
Figure 6:
Temperature dependence of the rate
constant for the reactions of ferrous cytochrome b and Tet-Mb. The reactions of cytochrome b
with ferricyanide (closed square) and
O
(closed circle) and the reaction of Tet-Mb with
O
(open circle) were measured at temperature
between 2 and 40 °C at pH 7.4. Initial concentrations of O
were 10 and 350 µM for the reactions of cytochrome b
and Tet-Mb, respectively, at 10 °C and
were corrected according to the standard solubilization curve for the
estimation of k
. The reactions of cytochrome b
with ferricyanide were measured under
experimental conditions as described in the legend to Fig. 5except temperature.
From the k value at 30 °C, the
oxidation rate of ferrous cytochrome b
by
O
is estimated at about 8000 s
under
air-saturated conditions, which is hundreds of times faster than the
rate of the steady state turnover, 1
20 s
in the
catalytic production of O
in situ or in our reconstituted system(13, 14) . The
reduction of ferric cytochrome b
under anaerobic
conditions was considerably slower ((13) ). (
)Furthermore, it is unlikely that a process following the
reduction of O
, such as product release, is rate-limiting
because the lifetime of O
, even in
proteins, should not be so long as the heme reduction. Thus, the
rate-limiting step in the catalytic reaction is the transfer of
NADPH-derived electrons to the heme, and the single turnover reaction
rather than the steady state one should be measured to investigate the
properties of the reaction of cytochrome b
with
O
or to examine the effects of respiratory inhibitors.
The rate of the oxidation of ferrous cytochrome b linearly depends on the O
concentration, indicating
that the reaction proceeds through a bimolecular process. During the
reaction, the spectra of only the two (ferrous and ferric) forms of
cytochrome b
could be detected, and no
intermediate species could be observed. This indicates that the
reaction intermediate, if any, transiently forms and is rapidly
decomposed to O
and the ferric heme.
Otherwise, the absorption spectrum of the intermediate is
indistinguishable from those of the ferrous and ferric forms under the
wavelength resolution. Neither the spectra of the ferric and ferrous
cytochrome nor the reaction with O
were affected by
addition of CO, CN
, and
N
. Artificial oxidizing agents,
ferricyanide and p-benzoquinone, can react with ferrous
cytochrome b
apparently through an outer sphere
mechanism. The rate of oxidation of ferrous cytochrome b
by ferricyanide was even larger than by
O
. The thermodynamic analyses show that the set of the
activation parameters for the reaction with ferricyanide is similar to
that for the reaction with O
, suggesting that the reaction
with O
occurs by a mechanism similar to that with
ferricyanide (Table 1, and also see below). The site for the
reaction with ferricyanide appears to be located at a solvent
accessible position on the protein and may be common with the site for
the reaction with O
. On the other hand, the site for the
reaction with p-benzoquinone may be different from that for
the reaction with O
and may be located at a position more
distant from the heme. The higher redox potential of ferricyanide may
contribute to the higher rate of the reaction. All of these results
suggest that the coordination of both the internal axial ligands to the
heme iron in cytochrome b
is so strong that
O
does not bind to the iron by replacing any internal
ligand during the reaction and that the reaction with O
proceeds via an outer sphere mechanism.
Support for our idea
mentioned above is provided by comparing the thermodynamic parameters
in the oxidation of cytochrome b by O
and ferricyanide with the reactions of cytochrome c and
Tet-Mb (Table 1). It was suggested that the reduction of ferric
cytochrome c by O
takes place at
or close to the solvent accessible heme edge on the basis of the
thermodynamic and kinetic studies(22) . On the other hand,
Tet-Mb, in which the distal histidine (His E7) of myoglobin is
site-specifically modified with tetrazole anion
(-CN
), can catalyze reduction of O
to O
in the presence of an
enzymatic reduction system(19) . Tet-Mb exhibits a low
oxidation reduction potential (E
=
-193 mV), which is comparable to that of cytochrome b
, and has the six-coordinated heme iron in the
ferric state by coordination of the tetrazole group as the internal
sixth ligand(20) . In contrast with cytochrome b
, the ferrous heme iron of Tet-Mb can bind CO
and forms an iron-O
complex in the reduction of
O
. Therefore, the mechanisms for the reactions of
cytochrome b
and Tet-Mb with O
seem
to mainly depend on the bond strength of the axial ligand to the sixth
coordination site of the heme iron.
In any of these reactions listed
in Table 1, the contributions of the entropic terms (TS
) to the total energy barriers (
G
) are small and the enthalpic
contributions (
H
) are predominant.
However,
H
is much more predominant in the
reaction by Tet-Mb than in those by cytochrome c and
cytochrome b
, whereas the T
S
values for the four reactions are
similar to each other. The large
H
value
for the reaction of Tet-Mb is similar to that for the autoxidation of
native myoglobin by the formation of the iron-O
complex
(85-150 kJ/mol)(23) . Since
H
is a measure of the energy barrier which must be overcome to
break and make bonds in the formation of the transition state from the
reactants, it seems reasonable that the lower enthalpic barriers in the
reactions by cytochrome c and cytochrome b
arise from the weaker interactions of O
with the ferric heme and of O
(or ferricyanide) with
the ferrous heme, respectively, in these transition states.
The
results obtained here, together with our previous
data(13, 14, 15) , are best understood
assuming an outer sphere mechanism for the reduction of O by cytochrome b
. The reaction can occur at
or near the heme edge by a ``peripheral-
-process,''
which has been proposed for the autoxidation of coordinately saturated
iron porphyrin complexes by Castro and
co-workers(24, 25) . In this process, O
weakly interacts with the porphyrin
plane of the ferrous
heme followed by the electron transfer. The weak interaction between
O
and the iron-porphyrin system is similar to those in
peripheral
complexes of metalloporphyrins with aromatic
compounds(26) . In the heme of cytochrome b
, the strong and symmetrical coordination of
the axial ligands can enhance the interaction between iron d and porphyrin
electrons to make the rapid O
reduction possible.
The weak interaction of O with
the heme of ferrous cytochrome b
may be
stabilized by hydrogen bonding with a basic amino acid residue near the
heme pocket since the reaction slows down by increase of pH above 9 (Fig. 4) as follows (Fig. S1).
Scheme 1:
In this scheme,
BH indicates the protonated basic residue. We have
obtained no spectroscopic evidence for such a loosely associated
complex of the heme with O
. This seems to be inevitable
since the formation of such a complex should be transient (half-time
<1 ms) and/or the weak interaction can induce only slight changes in
the absorption spectra that are not expected to be detectable under the
experimental conditions.
It is also possible that a positive charge
of an ionizable group near the heme rises the redox potential and gives
higher reactivity with O in the neutral pH range.
The
major type of oxidases and oxygenases contains five-coordinated heme
iron and catalyzes reduction of O bound to its sixth
coordination site to generate H
O or oxygenated products.
Some of these enzymes also catalyze production of
O
or H
O
in their
uncoupled or abnormal reactions (27, 28, 29) . In the present study, we have
suggested a novel mechanism for the reactions of the heme-containing
oxidases. The mechanism for the O
reduction may be common
in autoxidation of cytochromes with 6-coordinated hemes and also in
bimolecular reactions of deoxy myoglobin and hemoglobin with O
under specific conditions (30, 31, 32) . It appears that phagocytic
cytochrome b
has evolved the coordination
structure of the heme to be specifically adapted for the rapid
production of O
even under low
concentration of O
.