(Received for publication, March 27, 1995; and in revised form, April 6, 1995)
From the
Analysis of potentiometric titrations of the cytochrome b
Neutrophils contain a multicomponent, NADPH-dependent,
O
We previously showed that the Arg
Figure 1:
Potentiometric titration of wild-type
and Arg
Figure 2:
Potentiometric titration of wild-type and
Arg
From the data presented above, it appears that both wild-type
and mutant cytochromes contain two separate, nonidentical hemes. Both
contain a heme center with a midpoint potential around -220 mV
contributing approximately 50% to the total absorbance change; in the
wild-type cytochrome, there is a second, closely spaced center with E
It is
thought that invariant arginine residues near the membrane surface of
mitochondrial cytochromes b form hydrogen bonds with the
negatively charged propionyl groups of the heme (22 and references
therein). Hydropathy plots of gp91
In view of the fact that
the redox potential of the heme in cytochrome b
from a X
chronic
granulomatous disease patient with an Arg
Ser
mutation in gp91
indicates that the mutant form
of the cytochrome contains two nonidentical hemes with midpoint
potentials of E
= -220 and E
= -300 mV. In the light
of this information, reanalysis of redox titrations of wild-type
cytochrome b
implies that it probably
also contains two separate heme centers with midpoint potentials of E
= -225 and E
= -265 mV. The effect of
the Arg
Ser substitution is to reduce the midpoint
potential of one of the heme centers by approximately 35 mV and
suggests possible interaction between Arg
and a heme
propionate side chain.
generating system that is used as a
powerful antimicrobial weapon in the process of host defense (recently
reviewed in (1) ). The redox centers of this NADPH oxidase, FAD
and heme, are both contained within a unique low potential
flavocytochrome, cytochrome b
(also known
as cytochrome b
or cytochrome b
) that also contains the NADPH binding
domain(2, 3, 4) . Cytochrome b
is composed of a heavily glycosylated
large
-subunit (gp91
) and a small
-subunit (p22
), the products of two
separately regulated genes. Each subunit is apparently unstable in the
absence of its partner. The simplest model of cytochrome b
is that of a
gp91
/p22
heterodimer
containing 1 mol of heme and 1 mol of FAD. However, theoretical
considerations and experimental evidence from
purification(2, 5, 6, 7, 8) ,
proteolytic(9) , and reconstitution
(
)(10, 11) studies suggest that each
mole of cytochrome b
probably contains 2
mol of heme and 1 mol of FAD. In addition, optical, EPR, CD, and
resonance Raman spectra(7, 12, 13, 14, 15, 16) are
consistent with the presence of multiple, bis-histidinyl,
hexacoordinate, low spin hemes. Hitherto, the presence of only a single
species of heme in cytochrome b
has been
inferred from oxidation-reduction potentiometric studies, where the
cytochrome titrates as a single component with a midpoint potential of
-245 mV (17) . Here we present evidence from the studies
of the nonfunctional cytochrome from an X
CGD
(
)patient with an Arg
Ser mutation in gp91
that clearly
demonstrates the presence of two nonidentical hemes with midpoint
potentials of E
= -220 mV
and E
= -300 mV. In light
of this information, reanalysis of redox titrations of wild-type
cytochrome b
suggests the presence of two
hemes with closely spaced midpoint potentials of E
= -225 mV and E
= -265 mV.
Materials
Anthraquinone 2,6-disulfonate,
duroquinone, 2-hydroxy-1,4-naphthoquinone, and
2,3,5,6-tetramethylphenylenediamine were obtained from Aldrich.
Anthraquinone and sodium dithionite were supplied by Fluka and Fisher,
respectively; heptyl--D-thioglucopyranoside was from
Calbiochem. All other reagents were purchased from Sigma. Pyocyanine
was synthesized from phenazine methosulfate using the photochemical
method described in (18) .
Isolation of Neutrophils and Preparation of Subcellular
Fractions
Membranes and cytosolic fractions were prepared from
the unstimulated neutrophils of both normal and CGD donors as described
previously (19) and stored in aliquots at -80 °C.
Solubilization and Purification of Cytochrome
b
Cytochrome b was partially purified as described previously(20) .
Measurement of Oxidation-Reduction
Potentials
Potentiometric titrations were performed as described
previously (17) using partially purified cytochrome b preparations derived from 9
10
neutrophils in a total volume of 2.7 ml of 100 mM KCl, 50 mM MOPS, pH 7.0. The following mediators were
used at 12.5 µM: phenazine methosulfate, phenazine
ethosulfate, anthraquinone, anthraquinone 2-sulfonate, anthraquinone
2,6-disulfonate, 2-hydroxy-1,4-naphthoquinone,
2,3,5,6-tetramethylphenylenediamine. Pyocyanine was added at 6
µM. Spectra were recorded between 580 and 520 nm at a
series of electrode potentials, using a Uvikon 810 spectrophotometer.
The degree of reduction of cytochrome b
was estimated from the height of the
absorbance band. An
Orion 720A meter (Orion Research Inc., Boston MA) was used to measure
the half-cell potential relative to a saturated calomel reference
electrode. The potential was adjusted by the addition of <µl
volumes of solutions of sodium dithionite (reductive titrations) and
potassium ferricyanide (oxidative titrations). The accuracy of the
apparatus was checked by titration of a 5 µM solution of
phenosafranin (E
= -252
mV).
Ser
mutation in gp91
results in a cytochrome b
with a nonfunctional heme with a
slightly shifted Soret band that is unable to accept electrons from the
reduced flavin center(21) . In contrast, the flavin domain of
the cytochrome b
is fully functional, as
it is capable of accepting electrons from NADPH and reducing the
artificial dye acceptor iodonitrotetrazolium violet at rates equivalent
to that of the wild-type cytochrome. To determine if the loss of
function was due to an alteration in heme redox potential, we performed
a series of oxidation-reduction potential measurements using cytochrome b
partially purified from neutrophil
membranes from both normal and CGD patients. The results are shown in Fig. 1. The wild-type cytochrome titration could be fitted
fairly well (
) to a Nernst equation curve for a single
component with a E
= -245
mV (Fig. 1A), the same midpoint potential as originally
determined(17) . In contrast, titration of the Arg
Ser cytochrome clearly did not fit the expected simple
single component, 1-electron transfer process (Fig. 1B)
(
). By assuming two components are present, the
data could be fitted to curves corresponding to two species with
midpoint potentials of -220 and -300 mV, each contributing
50% to the absorbance at 559 nm (Fig. 2B) (
). By
inferring that two nonequivalent hemes are also present in the
wild-type cytochrome b
, the data in Fig. 1A can be fitted in a similar fashion to a
2-component curve with midpoint potentials of E
= -225 and E
= -265 mV (Fig. 2A) producing
an excellent fit (
). Titrations of the cytochrome b
from a CGD patient with a Pro
His mutation in gp91
in which the
flavin domain is nonfunctional (2, 21) were
indistinguishable from that of the wild-type cytochrome, in accordance
with a previous report (16) (data not shown).
Ser cytochrome b
; one component, 1-electron transfer.
Oxidation-reduction potential measurements were performed as described
under ``Experimental Procedures.'' Circles represent
oxidative titrations (potassium ferricyanide); squares,
reductive titrations (sodium dithionite). A, titration of
wild-type cytochrome b
; the solid
curve is a theoretical line for a single species, 1-electron
transfer process with a midpoint potential of -245 mV. B, titration of Arg
Ser cytochrome b; the solid curve is the theoretical curve for a
single species, 1-electron transfer with a midpoint of -260
mV.
Ser cytochrome b
;
two components, 1-electron transfer. A, data as in Fig. 1A with the solid curve representing two
species with midpoint potentials of -225 and -265 mV, each
contributing 50% to the total absorbance change. B, data as in Fig. 1B with the solid curve representing two
species with midpoint potentials of -220 and -300 mV, each
contributing 50% to the total absorbance change. Circles represent oxidative titrations; squares, reductive
titrations.
= -265 mV. The similarity
in redox potentials makes resolution of the two centers difficult in
the wild-type cytochrome. In the Arg
Ser
cytochrome, the potential of the second lower potential center has been
shifted downward by 35 mV from -265 mV to -300 mV and can
be clearly resolved. At the present time, the locations of the hemes
within the cytochrome are not known with any certainty.
p22
, which is reported to contain heme,
contains only one invariant histidine, the latter lying within a
sequence with homology to the heme-binding domains of a cytochrome
oxidase subunit and the chromaffin granule cytochrome b
. In view of the assignment of the axial
ligands in cytochrome b
as
bis-histidinyl, a single p22
subunit cannot
wholly contain a heme group. gp91
contains 17
histidines, none lying within sequences known to be homologous with
other heme proteins. Thus, one heme group is probably shared between a
p22
and gp91
subunit
(or two p22
subunits), and one heme is likely
to be contained within gp91
itself.
predict
Arg
to be at the beginning of a membrane-spanning segment
close to the membrane surface, and, therefore, this residue could
perform a similar hydrogen bonding function in cytochrome b
. The adjacent membrane-spanning segment
(amino acids 100-120) contains 3 potential heme-liganding
histidine residues. Substitution of the positively charged Arg
to an uncharged serine residue would decrease the electron
withdrawing nature of this group and thereby lower the redox potential
of the heme. Site-directed mutagenesis studies of the analogous
situation in iso-1-cytochrome c have demonstrated such
decreases in redox potential when glutamine (-30 mV), asparagine
(-34 mV), or alanine (-50 mV) were substituted for the
arginine (Arg
) that forms a hydrogen bond with one of the
heme propionate side chains(23) .
is altered by less than 40 mV by the Arg
Ser
substitution, it is perhaps surprising that the mutant form has no
detectable O
generating activity,
particularly since we have shown that the flavin center is fully
functional in this mutation(20, 21) . Further studies
using site-directed mutagenesis and other naturally occurring mutant
forms of cytochrome b
will be invaluable
in dissecting the factors affecting electron transfer functions in
cytochrome b
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.