From the
Cytochrome b
A phagocyte-specific cytochrome b
In the present study, we have further investigated the low
temperature ESR spectrum of cytochrome b
The ratio of the
high-spin state to the low-spin state of the heme in cytochrome
b
Cytochrome b
Under physiological conditions, the purified cytochrome
b
One of the important characteristics of low-spin
hemoproteins that exhibit highly anisotropic low spin-type ESR signals
with a low field component g
All the hemoproteins involved in
activation of oxygen, such as cytochrome c oxidase and
cytochrome P450, have a high-spin heme, in which the sixth coordination
site is open (or weakly coordinated by water) for binding oxygen. In
contrast, neutrophil cytochrome b
In conclusion, the results present that the low-spin
state of the heme in cytochrome b
purified from pig
neutrophils was studied to characterize the spin state of the heme iron
in relation to its O generating activity. ESR spectra of cytochrome
b
either from resting or stimulated neutrophils
showed a low-spin hemoprotein with g
of 3.2, 2.1, and
1.3 (estimated). At physiological pH, the oxidized cytochrome
b
is in a purely low-spin state. On lowering or
raising pH from 7, the spin state changes to high-spin. The ESR
spectrum of high-spin cytochrome b
was identical
to that of methemoglobin, suggesting that the axial-ligand type in both
hemoproteins may be the same, i.e. histidine is the fifth
ligand. The ratio of the low-spin to high-spin heme in cytochrome
b
was evaluated by magnetic circular dichroism
spectroscopy. The pH of cytochrome b
was varied
to form different ratios of the low-spin to high-spin states of the
heme, and its O generating activity was examined in cell-free systems.
O forming activity decreased concomitant with loss of the low-spin
heme, which provides direct evidence that the low-spin state of
cytochrome b
is essential to generate O and the
heme retains the low-spin state through the redox cycle.
is an
essential component of the membrane-bound NADPH oxidase system which
produces superoxide anion (O) in response to the invading
microorganisms
(1, 2) . Cytochrome b
is a heterodimer consisting of large and small subunits, both of
which have been sequenced
(3, 4) . The large subunit of
cytochrome b
is postulated to be a
membrane-bound flavocytochrome from its amino acid sequence homology
with other flavoproteins
(5, 6, 7) , affinity
labeling with NADPH analogues
(6, 8) , and binding of
FAD
(9) , but other flavoproteins, such as 77-kDa protein (10)
and nitro blue tetrazolium reductase
(11) , were also reported as
candidates for NADPH dehydrogenases. In spite of the flavocytochrome
hypothesis, no one has isolated an FAD-containing cytochrome
b
from neutrophil membranes. Cytochrome
b
has been postulated to function as the
terminal oxidase of the respiratory electron transfer chain due to its
unusually low redox potential (E
=
-245 mV)
(12) , CO-binding
capability
(12, 13, 14) , and rapid reoxidation
rate
(15, 16) . We carefully examined the CO-binding
capacity of cytochrome b
using solubilized
membrane fractions from either resting or stimulated cells, and
confirmed that the CO-binding capacity of undenaturated cytochrome
b
is very poor
(17) . Furthermore,
NADPH-dependent reduction of cytochrome b
in the
stimulated state of the oxidase is as slow as that in the resting
state, which may not explain the O generating activity of the NADPH
oxidase system
(18, 19) . Recently, we purified
cytochrome b
without losing its O forming
activity in a cell-free system
(11) and succeeded in assigning
the ESR signal at g value of 3.2 to the hexa-coordinated low-spin heme
iron of cytochrome b
(17, 20) .
, which
was purified and concentrated without denaturation from resting and
stimulated neutrophils. The results confirm that both preparations of
cytochrome b
exhibit a rhombic ESR spectrum with
g values of 3.2, 2.1, and 1.3, similar to that of mitochondrial
cytochrome b(21) . In addition, the spin state of the
heme iron was modified by varying the pH of cytochrome
b
preparations to yield samples with perturbed
high-spin to low-spin ratios. Using these preparations, we studied the
correlation of the O generating activity with spin state of these
cytochrome b
in order to clarify whether the
low-spin state of cytochrome b
is essential to O
generation in the NADPH oxidase system.
Materials
DEAE-Sepharose CL-6B and
heparin-Sepharose 6B were obtained from Pharmacia LKB Biotechnology
Inc. n-Heptyl--thioglucoside and EGTA were purchased from
Dojindo Laboratories, Kumamoto. NADPH was purchased from Oriental
Yeast, Tokyo. Sodium myristate and diisopropyl fluorophosphate were
from Wako Pure Chemicals Co., Tokyo. Superoxide dismutase, cytochrome
c (type VI, from horse heart), and human hemoglobin were
purchased from Sigma. GTP
S
(
)
was a product
of Boehringer Mannheim. Other chemical reagents were of analytical
grade.
Purification of Cytochrome b
Resting or stimulated
neutrophils were obtained from pig blood as described previously
(22) and treated with 2 mM diisopropyl fluorophosphate
for 20 min at 0 °C. The membrane fraction was obtained from the
sonicated neutrophils, and then cytochrome bfrom Pig Blood Neutrophils
was
solubilized from the membrane fraction with
n-heptyl-
-thioglucoside according to the previous method
(18). Cytochrome b
was purified from solubilized
membranes according to the method reported
previously
(11, 20) , except that a heparin-Sepharose
column was equilibrated with a buffer composed of 50 mM
phosphate buffer (pH 7.0), 50 mM NaCl, 10% glycerol, and 0.6%
n-heptyl-
-thioglucoside.
Assay of Cell-free O Production
The O generating
activity of the NADPH oxidase was determined at 25 °C in a
reconstituted cell-free assay system according to a reported method
(11) with slight modification. The pH of the purified cytochrome
b preparation was adjusted to a desired pH by
mixing with small amounts of 0.5 M NaOH or 0.5 M HCl,
and the preparations were kept for 10 min on ice. Then, the pH of such
a preparation was readjusted to 7.0 by HCl or NaOH. This cytochrome
b
preparation was reconstituted by incubating
with an appropriate amount of the nitro blue tetrazolium reductase
fraction
(11) for 30 min on ice. Thereafter, the mixture
containing 2 pmol of cytochrome b
was incubated
for 5 min at 25 °C with cytosol from resting cells, 20
µM GTP
S, and an appropriate amount of sodium
myristate in the assay medium composed of 50 mM phosphate
buffer (pH 7.0), 2 mM NaN
, 1 mM EGTA, and
1 mM MgCl
, and then the assay medium containing 50
µM cytochrome c was added to the above mixture to
give a final volume 0.8 ml. O production was measured with a Hitachi
dual-wavelength spectrophotometer (model 556) by recording the
reduction of cytochrome c at 550 nm after addition of 125
µM NADPH.
Spectrophotometric Measurements
Absorption spectra
were measured at 25 °C in the range of 400 and 600 nm in a
microcuvette (10-mm light path, 3-mm width) in a Unisoku
Biospectrophotometer US-401 (Unisoku Co. Ltd., Osaka), controlled by
the computer, NEC PC9801. The volume of the sample used in the
microcuvette was 120-150 µl. MCD spectra were recorded using
JASCO J-500 spectropolarimeter interfaced to a Jasco MCD-1B
electromagnet. Spectra were recorded in the wavelength range from 300
to 800 nm, at a magnetic field of 1 tesla and at a temperature of 25
°C.
ESR Spectra
ESR spectra were recorded in a Jeol
(JES-FE) X-band ESR spectrometer equipped with LTR Heli-Tran liquid
helium transfer refrigerator (Air Products and Chemicals Inc.). The
conditions for measurements were as follows: microwave power,
0.16-10 mW; modulation amplitude, 10 G at 100 KHz; response, 0.3
s; sweep time, 4 min; temperature, 5 to 20 K. Copper EDTA in water was
employed as a concentration standard. For the measurement of ESR
spectrometry, both cytochrome b-rich membranes
and the purified cytochrome b
were concentrated
with Amicon Centricon-30 at 5 °C. The pH of the concentrated sample
was adjusted by aliquots of 0.5 M NaOH or 0.5 M HCl
at 0 °C, and was measured by Cosmo pH Boy-C1 at 25 °C.
Heme Environment of Oxidized Cytochrome
b
Cytochrome b was purified
from pig blood neutrophils without any denaturation of the heme
environment, since the cell-free system employing the purified
cytochrome b
exhibited high O generating
activity. The catalytic activity of the cell-free systems with the
purified cytochrome b
was 55-63 mol of O
produced per s/mol of cytochrome b
, which was
more than 80% of that of the solubilized membranes, 73 mol of O. The
ESR spectrum of the purified cytochrome b
prepared from resting neutrophils was measured at 5-20 K,
in which a spectrum recorded at 10 K was shown in Fig. 1. No
difference in the spectrum was found in purified cytochrome
b
prepared from the stimulated neutrophils.
Cytochrome b
in the oxidized state at pH 7.0
showed a low-spin ESR spectrum with g value of 3.2 in accord with that
of the previous reports
(17, 20) , but the present
preparation showed much weaker ESR signals from unknown origins at g
values of 6, 4.3, and 2, as compared with the previous
signals
(20) . In the previous report
(20) , only the low
field component at g = 3.2 was assigned. The improved ESR data,
shown in Fig. 1, clearly show the associated derivative component
at g
2.1. On the basis of these two g values, the high field g
value is expected near g = 1.3. However, in common with other
hemes with similar g value anisotropy, this feature is broad and
difficult to discern above the background. The g value anisotropy is
not as large as that encountered for mitochondrial cytochrome b exhibiting highly anisotropic low spin-type signals (g
= 3.4-3.8), but in common with these species the
low-spin resonance of cytochrome b
is fast
relaxing and requires low temperature (5-20 K) and high microwave
powers (10-20 mW) to facilitate observation.
Figure 1:
ESR spectrum of cytochrome
b recorded at 10 K. The purified cytochrome
b
was concentrated to 103 µM and
the pH in the medium was adjusted to 7.0 with 50 mM phosphate
buffer. The ESR instrumental settings were as follows: microwave
frequency, 9.04 GHz; microwave power, 10 mW; modulation amplitude, 10
gauss; response time, 0.3 s.
The effect of pH
on the absorption spectra of purified cytochrome b was studied at 25 °C, and the series of dithionite-reduced
minus oxidized difference spectra measured at different pH are
illustrated in Fig. 2. Difference spectra at around physiological
pH, having a
-band at 428 nm, a
-band at 530 nm, and an
-band at 558 nm, are characteristic of the low-spin heme. On
lowering pH from 7 to 3.1, however, the difference spectra were
broadened with concomitant loss of the signal intensities at the
-,
-, and
-bands. Below around pH 4, the
-peak was
remarkably broadened, followed by the increase in the shoulder at
around 437 nm. In contrast to the acidic region, the remarkable change
in difference spectra was not seen in alkaline medium until around pH
11, but a slight loss of the signal intensities at the
-,
-,
and
-bands was detected. The absorption spectra in
Fig. 2
indicate that the pH in the cytochrome b
preparation induces a change in the spin state of the heme in
both acidic and alkaline medium.
Figure 2:
Effect of pH on the reduced minus oxidized
absorption difference spectra of cytochrome b at
25 °C. The pH in the samples were (from top to bottom): pH 11.6,
10.3, 9.2, 7.0, 5.0, 4.1, and 3.1. Purified cytochrome
b
was diluted with 50 mM phosphate
buffer (pH 7.0) containing 0.6% n-heptyl-
-thioglucoside
to a final concentration of 2.1 µM. The pH in the
cytochrome b
preparation was adjusted by adding
aliquots of either 0.5 M NaOH or 0.5 M
HCl.
The effect of pH on the heme
environment of cytochrome b was studied using
ESR spectrometry. ESR spectra of oxidized cytochrome b
in the pH region from 4.1 to 11.8 are shown in Fig. 3. The
ESR spectrum at pH 7 is dominated by the g = 3.2 feature of the
low-spin hemoprotein (Fig. 3). On lowering or raising the pH from
7, new ESR signals appeared at g = 6.0 under acidic pH and at a
g value of 6.2 under alkaline pH. Both signals are typical of high-spin
ferric hemes, and their appearance occurs concomitant with the
disappearance of the low-spin signal at g = 3.2. The change in
ESR spectra in either acidic or alkaline pH was irreversible. The
spin-state transition of the heme from low-spin to high-spin was more
pronounced to acidic pH values. At alkaline pH, a new low-spin ESR
signal appears, g
= 2.42, 2.23, and 1.91, in
addition to the high-spin signal at g = 6.2. This most likely
reflects an equilibrium between a five-coordinate high-spin heme and a
six-coordinate low-spin heme with altered axial ligation. Fig. 4,
A and B, illustrates the comparison of the ESR
spectrum of cytochrome b
at pH 3.5 with that of
methemoglobin at pH 7.0. As shown in Fig. 4A, at acidic
pH all of the low-spin heme signals at g = 3.2 disappeared and
the low-spin heme has been completely converted to a S = 5/2
high-spin heme with g
= 6.0 and g
= 2.0. The ESR spectrum of methemoglobin at pH 7, a
typical high-spin heme, is shown in Fig. 4B, and is
identical with that of the high-spin cytochrome b
at pH 3.5 (Fig. 4A). This suggests that the heme
environment of cytochrome b
in the high-spin
state is remarkably similar to that of hemoglobin, which in turn
implicates histidine as the fifth ligand. The binding of cyanide to the
high-spin heme iron in cytochrome b
was
confirmed by ESR spectroscopy (data not shown), and the CO binding was
also confirmed by reduced-minus-oxidized difference spectra, showing
shift in its
-band from 428 to 420 nm upon binding CO.
Figure 3:
Effect
of the pH on the ESR spectra of purified cytochrome b. The
purified cytochrome b was concentrated to be 85
µM, and then the pH was adjusted as in Fig. 1. The pH
values are as follows (from top to bottom): pH 11.8, 10.2, 7.0, 5.0,
and 4.1. The instrumental conditions for all spectra were identical to
those in Fig. 1, except that the microwave power was set as 1 mW.
Receiver gains were scaled as indicated on the
figure.
Figure 4:
Comparison of ESR spectrum of the
high-spin cytochrome b (A) with that of
hemoglobin (B). The purified cytochrome b
was concentrated to 95 µM, and the pH value in the
preparation was adjusted to 3.5, as described in the legend to Fig. 3.
Methemoglobin was dissolved into 50 mM phosphate buffer (pH
7). The concentration of methemoglobin used for ESR measurements was
125 µM. The instrumental conditions for all spectra were
identical to those in Fig. 1, except microwave power = 0.16
mW.
The ESR
signal associated with the low-spin ferric heme in cytochrome
b, see Fig. 1, is too broad for accurate
spin quantitation. Moreover it is overlapped by other ill-defined
signals particularly in the g = 2 region. In contrast, as shown
in Fig. 4, the high-spin ESR signal that appears at acidic pH is
much more intense and is amenable to spin quantitation by double
integration versus a Cu
standard under
non-saturating conditions. At 10 K, this procedure indicated a spin
quantitation of 88 µM. However, this will be an
underestimate since the g
= 6.0 and g
= 2.0 resonance originates from the lowest
(M
= ±1/2) doublet ground
state manifold and no attempt was made to allow for population of
M
= ±3/2 or ± 5/2
doublets. Nevertheless this is close to the value of 95 µM
which was estimated by optical spectrometry based on a
reduced-minus-oxidized extinction coefficient (558-540 nm) of
21.6
10
liter mol
for cytochrome
b
(23) . Hence the low pH high-spin ESR
signal appears to account for all of heme.
was estimated by MCD spectrometry. The MCD
spectra in the region 300-800 nm were recorded at 25 °C of
purified cytochrome b
in the oxidized state as a
function of pH. At room temperature the peak-to-trough Soret band MCD
intensity of low-spin ferric hemes is at least 20 times greater than
that of high-spin ferric hemes
(24) . Hence the percentage of
low-spin heme as a function of pH can be conveniently assessed based on
the peak-to-trough Soret band intensity. On the basis of the ESR data,
the pH 7 data point was taken as 100%. Percent of the low-spin heme in
cytochrome b
obtained by MCD spectrometry is
plotted against pH in Fig. 5.
Figure 5:
Effect of
pH on percent of the low-spin state of the heme () in cytochrome
b
and O generating activity (
) of NADPH
oxidase system. pH in purified cytochrome b
was
adjusted to form various different ratios of the low-spin to high-spin
heme. The MCD spectra in the region 300-800 nm were recorded at
25 °C of these purified cytochrome b
in the
oxidized state. Percent of the low-spin state of the heme in cytochrome
b
was calculated by the intensity of the Soret
band MCD. The O generating activity of the purified cytochrome
b
with different ratios of the low-spin to
high-spin heme was measured in a reconstituted cell-free
system.
O-generating Activity and the Spin
State
To clarify the relationship between the spin state of
the heme in cytochrome b and the O generating
activity of the NADPH oxidase system, the O generating activity was
measured in a reconstituted cell-free system consisting of the purified
cytochrome b
with different ratios of the
low-spin to high-spin heme prepared by changing the pH, as in
Fig. 3
. Fig. 5shows the effect of the functional spin
state of the heme in cytochrome b
on the O
generating activity of the NADPH oxidase system. The optimum O
generating activity was found in the vicinity of pH 7, which was the
same as that observed previously in the stimulated membrane
system
(25) . On lowering pH in cytochrome
b
, the O generating activity of the NADPH
oxidase system decreased concomitant with the decrease in percent of
the low-spin heme in cytochrome b
. These results
indicate that cytochrome b
is in the low-spin
state when the NADPH oxidase system produces O in the cell-free system.
In order to further confirm that cytochrome b
is
in the low-spin state when the oxidase system is generating O, ESR
spectra of cytochrome b
-rich membranes
solubilized both from resting and stimulated neutrophils were measured
in the presence of NADPH. The spectra are shown in Fig. 6, and
indicate that cytochrome b
in both preparations
is predominantly in the low-spin state. These results provide evidence
that the low-spin state of the heme in cytochrome b
is essential to generate O in the activated state of the NADPH
oxidase system.
Figure 6:
The
ESR spectra of cytochrome b-rich membranes
prepared from stimulated (A) and resting (B)
neutrophils. Cytochrome b
-rich membranes were
prepared by concentrating the NADPH oxidase obtained both from
stimulated and resting neutrophils. The concentrations of the heme in
both preparations were 48 µM. After incubating cytochrome
b
-rich membranes with 200 µM NADPH
in 50 mM phosphate buffer (pH 7.0) at 25 °C for 1 min, the
mixture in the ESR tube was rapidly frozen.
was purified from pig
blood neutrophils without any denaturation of the heme environment,
since the purified cytochrome b
exhibited high O
generating activity in the cell-free system. These purified
preparations had greatly decreased resonances at g = 6.0, 4.3,
and 2.0, compared with earlier reports
(17, 20) . The
purified cytochrome b
prepared in this study
exhibited the rhombic ESR spectrum with g
= 3.2,
2.1, and 1.3 (estimated), in which the g
component is much
broader and less pronounced than the g
component.
Biochemically important hemoproteins found in several
electron-transport chains, such as cytochrome b
and cytochrome b
in
mitochondria
(26, 27) , exhibit a low field g value which
is substantially larger than 3.1, and other g values, g
and
g
, are not readily observed, since the g
signal
is much broader and less intense than the g
signal. From
the ESR spectrum in Fig. 1, the heme environment of neutrophil
cytochrome b
seems to resemble that of
bis-histidine b-type hemoproteins found in the mitochondrial
electron-transport chain
(21, 27, 28) . Low
temperature near-IR MCD studies will provide a more definitive
assessment of the axial ligation and such studies are in progress.
is in the low-spin state, but at acidic pH the
low-spin heme transformed to the high-spin heme, as evidenced by the
characteristic high-spin ESR signal at g = 6. The ESR signal at
g = 6, which appeared at the acidic pH, was previously thought
to originate from other hemoproteins such as hemoglobin contaminated
into the purified cytochrome b
(20) , but
the present study indicates it to be due to a high-spin heme from
denaturated cytochrome b
. On the other hand, at
alkaline pH the low-spin heme is converted to a mixture of high-spin
heme and a different low-spin heme with g
=
2.42, 2.23, and 1.91. This low-spin ESR signal is very similar to that
of cytochrome P450 (29, 30), and a similar signal is also observed in
pyridine-treated cytochrome b
(31) . This
suggests that alkaline pH induces a structural modification in the heme
environment of cytochrome b
by shifting the
fifth heme ligand, histidine, to a nearby thiolate group of cysteine
residue. The sixth ligand is presumably hydroxide. This low-spin form
was unable to support O generation in the NADPH oxidase
system
(31) . An alternative candidate for this ESR signal is a
low-spin hydroxide adduct with histidyl and hydroxide axial ligation.
While we cannot rule out this explanation, the g value anisotropy is
more indicative of a species with cysteinate and hydroxide axial
ligation.
3.2 is that the ESR signal intensity
or the transition probability at the g
signal is very small
relative to low-spin hemes with g
between 2.4 and
2.9
(26) . Hurst et al.(32) failed to observe
the ESR signal of cytochrome b
in solubilized
neutrophil membranes, although their instrument has the sensitivity to
detect ESR signals of a typical low-spin hemoprotein, cytochrome
c, when examined at similar concentrations. These authors
explained the absence of a low-spin ESR signal of cytochrome
b
by the relaxation-induced signal broadening
due to a strong heme-heme interaction. In light of the present work,
such explanations are unnecessary and it would appear that cytochrome
b
contains a magnetically-isolated
b-type cytochrome.
has been shown
to contain a low-spin six-coordinate hemoprotein from ESR and
spectrophotometric
studies
(15, 16, 17, 18, 19, 20, 23, 32) .
By correlating O production with the percent of low-spin cytochrome
b
in samples of varying pH, we have shown that
the heme is low-spin during the production of O. Furthermore, transient
high-spin heme could not be detected, when cytochrome
b
-rich membranes prepared from stimulated
neutrophils were incubated with NADPH. Both results provide evidence
that cytochrome b
is in the low-spin state while
the oxidase system is forming O. Cytochrome b
was originally postulated to be the terminal enzyme of the NADPH
oxidase system from its CO-binding capability
(12) and unusual
low redox potential
(12) . Yamaguchi et al.(14) reported that purified cytochrome b
had a capability to bind CO. However, only high-spin heme iron of
cytochrome b
, i.e. denaturated form of
the heme iron, was found to be capable of binding both CO and cyanide.
On the other hand, Isogai et al.(16) reported that the
ferrous cytochrome b
was rapidly oxidized by
O
and concluded that an electron is directly transferred
from the ferrous heme to O
without ligation of O
to the heme iron. If O is generated directly from the reduced
heme in cytochrome b
, some electron acceptors,
such as menadione and quinone, might inhibit the electron flow to
O
. However, these electron acceptors showed no effect on
the O generating activity, indicating that all the electrons from NADPH
were selectively transferred to O
(18, 33) .
More detailed studies on the mechanisms of the electron transfer
reactions in hexa-coordinated low-spin hemoproteins are clearly called
for. The alternative possibility that O is not produced directly from
the heme in cytochrome b
also has to be
considered.
is essential
to generate O in the NADPH oxidase system, and that any transient
high-spin heme in cytochrome b
does not
contribute to the O generating activity.
S, guanosine 5`-(
-thio)triphosphate; MCD,
magnetic circular dichroism spectroscopy; mW, milliwatt.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.