(Received for publication, October 17, 1994; and in revised form, November 28, 1994)
From the
The effect of pyridine on the heme environment of cytochrome b was studied using ESR and optical absorption
spectroscopy in relation to the
O
-generating activity in the NADPH
oxidase system of stimulated pig neutrophils. As the concentration of
pyridine increased, the absorption maxima of the
- and
-bands
of cytochrome b
shifted which correlated with a
concomitant decrease in O
-generating
activity. In addition, the g = 3.2 signal of cytochrome b
decreased with the concomitant appearance of a
new ESR spectrum that strikingly resembled that of cytochrome P450. The
results suggest that pyridine induces a structural modification in the
heme environment of cytochrome b
by shifting the
5th heme ligand (histidine) to a nearby thiolate group without direct
binding of pyridine to the heme. The existence of a reactive thiolate
near the heme iron was confirmed by pretreatment of blocked cytochrome b
with p-chloromercuribenzoate, which
completely inhibited the formation of the cytochrome P450-like ESR
spectrum. The results provide further evidence that a low-spin heme
iron of cytochrome b
with a g-value of 3.2 is
essential to the O
-forming reaction of
the NADPH oxidase system. From sequence alignments of cytochrome P450
with those of large and small subunits of cytochrome b
, the heme in cytochrome b
appears to be specifically associated with the large subunit.
A phagocyte-specific cytochrome b is an
essential component of the membrane-bound NADPH oxidase system which
produces superoxide anions (O
) in
response to invading microorganisms (for reviews, see (1) and (2) ). The functional importance of the phagocytic NADPH
oxidase as well as the critical role of cytochrome b
were exemplified from studies of chronic granulomatous disease (3, 4, 5) from extensive detailed
immunological and biological studies of cytochrome b
(6, 7, 8) .
Cytochrome b is a membrane-bound heterodimer
of a 91-kDa glycoprotein (6, 7) and a 22-kDa
subunit(7, 8) . The amino acid sequences of the large
and small subunits of cytochrome b
were
determined from the corresponding cDNA (7) . Although detailed
primary structure data on cytochrome b
have been
reported, the location and the function of the heme iron have not been
characterized as well. Cytochrome b
was thought
to behave like the terminal component of an electron transport chain in
the NADPH oxidase system because of its low redox potential (9) and from spectroscopic studies(10, 11) ,
but this has not been proven directly. Pyridine has been shown to
inhibit both the respiratory burst in intact neutrophils and
NADPH-dependent oxygen consumption in lysed cells(12) . The
inhibition was accompanied by a spectral change in reduced cytochrome b
at 77 K, suggesting that pyridine binds to the
heme iron(12) . However, recently it was reported that pyridine
was not an inhibitor of oxygen consumption in a model
O
-forming system consisting of purified
cytochrome b
plus an exogenous
flavoenzyme(13) . After considering these contrary reports, it
became clear that it was important to confirm whether pyridine binds
directly to the heme iron of cytochrome b
, and
if it inhibited the NADPH oxidase system in neutrophils.
Recently,
we assigned the g = 3.2 ESR signal of the low-spin heme iron of
cytochrome b(14, 15) , which
was similar to the mitochondrial b-type cytochromes. In this
study, we further examine the effects of pyridine on the
O
-forming activity of the NADPH oxidase
from solubilized membranes of stimulated neutrophils in relation to
modifications of the heme environment of cytochrome b
using ESR and absorption spectroscopy. We observed a
dose-dependent inhibitory effect of pyridine on the oxidase activity
which was precisely linked to both visible and ESR spectral changes,
suggesting that pyridine induced a modification of the heme
environment, but without binding directly to the heme iron.
Unexpectedly, we found a new ESR signal in pyridine-treated cytochrome b
, which was very similar to that of cytochrome
P450, providing a clue to the location of the heme.
Fig. 1shows the inhibitory effects of pyridine on the
O-generating activity of solubilized
NADPH oxidase from stimulated neutrophils with time of incubation. An
inhibition of O
-forming activity was
observed with increasing pyridine concentrations, which reached to a
plateau after a relatively short incubation time, 10 min. (50%
inhibition at 30-50 mM pyridine after 10 min
incubation.) Relatively high concentrations of pyridine were required
to inhibit the O
-generating activity
effectively, suggesting that the effect was possibly due to a
structural modification of cytochrome b
, rather
than the simple binding of pyridine to the heme iron.
Figure 1:
Time course of the inhibitory effect of
pyridine on the O-generating activity of
the solubilized NADPH oxidase prepared from stimulated cells. NADPH
oxidase was incubated over varying time periods in the presence of
increasing concentrations of pyridine, prior to the
O
-generating activity assay. The
concentrations of pyridine used were 12.5 mM (a,
), 75 mM (b,
), 125 mM (c,
), and 150 mM (d,
).
Absorption
spectra of the solubilized oxidase were measured in the presence of
increasing pyridine at 25 °C. Difference spectra of
dithionite-reduced minus oxidized oxidase are shown in Fig. 2.
In the absence of pyridine, cytochrome b of the
solubilized oxidase shows
- and
-bands at 559 and 427 nm,
where no remarkable changes were observed up to 50 mM pyridine (trace c in Fig. 2). Upon increasing the pyridine
concentration up to 500 mM, the peaks shifted to 556 and 422
nm, respectively, accompanied by a decrease in peak intensities (traces d and e in Fig. 2). At 50 mM pyridine (Fig. 2, trace c), a slight shift occurs
in the
-band. Prolonged incubation (over 10 min) produced no
additional changes in the difference spectra, which was similar to that
observed for the inhibition by pyridine on
O
-forming NADPH oxidase activity (Fig. 1).
Figure 2:
Effect of pyridine on the difference
spectra of cytochrome b in the solubilized NADPH
oxidase. Solubilized oxidase (30 µg of protein) was preincubated
for 10 min in 50 mM phosphate buffer, pH 7.0, containing
varying amounts of pyridine: 0 (trace a), 10 (trace
b), 50 (trace c), 100 (trace d), and 500 mM (trace e). Solid dithionite was added prior to measuring
the difference spectra.
Since at quite high concentrations of pyridine, the
changes in the absorption spectra of cytochrome b were very small, we investigated if ESR spectrometry could yield
information on the spin state and its environment of the heme iron. ESR
spectra of solubilized oxidase (corresponding to 48 µM cytochrome b
) were measured with increasing
pyridine concentration at 10 K (Fig. 3). In the absence of
pyridine, the g = 3.2 ESR signal, assigned to the low-spin heme
of cytochrome b
, was prominent (Fig. 3, trace a)(14, 15) . Another low-spin ESR
signal was also observed at g = 3.0(14) , which was
tentatively assigned to a different state of cytochrome b
. The g = 3.2 signal decreased with the
concomitant appearance of new ESR signals at g-values of 1.9-2.4,
when the concentration of pyridine was increased to 0.5 M (Fig. 3, trace d). (Note that the pH of the sample
unchanged by pyridine addition.) The new ESR signals were strikingly
similar to those reported for cytochrome
P450(20, 21) . In order to confirm that the new ESR
signals in Fig. 3, trace d, were attributable to a
pyridine-modified form of cytochrome b
, purified
cytochrome b
was measured at various
concentrations of pyridine. Fig. 4, traces A and B, show typical ESR for 65 µM cytochrome b
in the absence and presence of 0.5 M pyridine, respectively. In the absence of pyridine, only the g
= 3.2 signal, but not the g = 3.0 signal, was observed (Fig. 4, trace A). In the presence of pyridine, the g
= 3.2 signal disappeared and new ESR signals appeared at
g-values of 2.36, 2.25, and 1.91 (Fig. 4, trace B),
which were similar to those as seen in Fig. 3, trace d.
Figure 3:
Effect of pyridine on the ESR spectra of
cytochrome b in solubilized oxidase prepared
from stimulated cells. ESR spectra of the concentrated solubilized
oxidase containing 48 µM cytochrome b
measured at 10 K in the presence of various concentrations of
pyridine: 0 (trace a), 100 (trace b), 200 (trace
c), and 500 mM (trace d). ESR instrumental
conditions were: microwave power, 10 mW; modulation, 6.3 G at 100 KHz;
response time, 0.3 s.
Figure 4:
Effect of pyridine on the ESR spectra of
purified cytochrome b in the absence and
presence of PCMB. Purified cytochrome b
(65
µM) was first measured without pyridine (trace
A). Then the same preparation was incubated for 10 min in the
absence (trace B) or presence (trace C) of 2 mM PCMB followed by addition of 0.5 M pyridine. ESR
instrumental conditions were identical to those in Fig. 3.
The results shown above suggest that the heme structure in
cytochrome b is modulated by pyridine and
becomes similar to that of cytochrome P450, suggesting that the 5th
ligand of the heme in pyridine-modified cytochrome b
has been replaced with a thiolate group present in the vicinity
of the heme. In order to examine whether such a reactive thiolate group
also exists in cytochrome b
, the sulfhydryl
reagent, PCMB, was employed as a blocking for reactive thiolate
cysteine groups. Purified cytochrome b
was first
treated with 2 mM PCMB, then mixed with 0.5 M pyridine and measured by ESR at 10 K. The resultant spectrum,
depicted in Fig. 4, trace C, shows that
``cytochrome P450-like'' signals induced by pyridine were
missing after PCMB treatment and a high-spin ESR signal appeared at g
= 6.
We have shown that pyridine causes shift in the -band
from 559 to 556 nm (Fig. 2) in agreement with the results
reported by Iizuka et al.(12) using low temperature
spectroscopy. In their report, inhibition of the
O
-generating activity by pyridine was
explained by the displacement of the internal heme ligands by a
pyridine molecule resulting in changes in the oxidation-reduction
potential of cytochrome b
(12) . However,
relatively high concentrations of pyridine were required to inhibit the
O
-generating activity, and no release of
hemin was observed. This possibly suggests that the pyridine inhibitory
effect is due to a structural (conformational) modification of
cytochrome b
, instead of a displacement of the
internal heme ligands with pyridine.
Scheme I: Scheme 1
Last, our previous studies
using ESR spin labels showed that the
O-releasing site was not located at the
outer surface of the neutrophil plasma membrane, but instead in an
interior hydrophobic environment, a short distance from the outer
surface(29) . Two potential heme iron ligands, namely
His
and His
, are located at an interior
hydrophobic region close to the glycosylation sites (Asn
,
Asn
, and Asn
) of the large
subunit(30) .