(Received for publication, August 7, 1995; and in revised form, September 14, 1995)
From the
Site-directed mutagenesis of the acidic clusters Asp-Asp-Asp
and
Glu-Glu-Asp
of NADPH-cytochrome P450
oxidoreductase demonstrates that both cytochrome c and
cytochrome P450 interact with this region; however, the sites and
mechanisms of interaction of the two substrates are clearly distinct.
Substitutions in the first acidic cluster did not affect cytochrome c or ferricyanide reductase activity, but substitution of
asparagine for aspartate at position 208 reduced cytochrome
P450-dependent benzphetamine N-demethylase activity by 63%
with no effect on K
or K
. Substitutions in the second acidic
cluster affected cytochrome c reduction but not benzphetamine N-demethylase or ferricyanide reductase activity. The E213Q
enzyme exhibited a 59% reduction in cytochrome c reductase
activity and a 47% reduction in K
under standard conditions (
0.27 M potassium
phosphate, pH 7.7), as well as a decreased K
at every ionic strength and a shift of the salt dependence of
cytochrome c reductase activity toward lower ionic strengths.
The E214Q substitution did not affect cytochrome c reductase
activity under standard conditions, but shifted the salt dependence of
cytochrome c reductase activity toward higher ionic strengths.
Measurements of the effect of ionic strength on steady-state kinetic
properties indicated that increasing ionic strength destabilized the
reductase-cytochrome c
ground state and
reductase-cytochrome c transition state complexes for the
wild-type, E213Q, and E214Q enzymes, suggesting the presence of
electrostatic interactions involving Glu
and Glu
as well as additional residues outside this region. The ionic
strength dependence of k
/K
for the
wild-type and E214Q enzymes is consistent with the presence of
charge-pairing interactions in the transition state and removal of a
weak ionic interaction in the reductase-cytochrome c transition-state complex by the E214Q substitution. The ionic
strength dependence of the E213Q enzyme, however, is not consistent
with a simple electrostatic model. Effects of ionic strength on kinetic
properties of E213Q suggest that substitution of glutamine stabilizes
the reductase-cytochrome c
ground-state
complex, leading to a net increase in activation energy and decrease in k
. Glu
is also involved in a
repulsive interaction with cytochrome c
.
Cytochrome c
K
for the wild-type enzyme was 82.4 µM at 118
mM ionic strength and 10.8 µM at 749 mM ionic strength; similar values were observed for the E214Q enzyme.
Cytochrome c K
for the E213Q enzyme was
17.6 µM at 118 mM and 15.7 µM at 749
mM ionic strength, consistent with removal of an electrostatic
repulsion between the reductase and cytochrome c
.
The microsomal and nuclear envelope flavoprotein
NADPH-cytochrome P450 oxidoreductase (P450R) ()(NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4)
catalyzes electron transfer from NADPH to the cytochromes P450 (1) and other microsomal
proteins(2, 3, 4) , as well as to
nonphysiologic electron acceptors such as cytochrome c,
ferricyanide, menadione, and
dichlorophenolindophenol(5, 6) . There is a
substantial body of information on the structure and mechanism of this
multidomain enzyme (7, 8) , as well as on its gene
structure and regulation(8, 9, 10) . Although
crystals of P450R have been obtained(11) , the crystal
structure has not been solved. FMN, FAD, and NADPH-binding domains of
P450R have been identified by sequence comparisons with flavoproteins
of known three-dimensional structure (8, 12, 13) and site-directed mutagenesis has
identified amino acids necessary for binding of FMN and
NADPH(14, 15, 16) . The orientation of the
nicotinamide and FAD isoalloxazine rings has been shown to be exo, with transfer of the pro-R hydrogen of NADPH to
FAD(17) . The kinetic mechanism with the substrate cytochrome c is nonclassical (two-site) Ping Pong, with the reaction of
cytochrome c at the electron acceptor site being Ping Pong at
high ionic strength and random sequential at low ionic
strength(18, 19) .
Less is known about the
substrate binding sites of P450R. A number of electron-transfer
complexes are stabilized by electrostatic interactions which play a
role both in complex formation and in orienting the two redox
centers(20, 21, 22) ; however, evidence also
exists for involvement of multiple hydrophobic and van der Waals
interactions (23, 24, 25) . Chemical
modification and cross-linking studies (26, 27, 28, 29) have provided
evidence for electrostatic interactions between P450R and its
substrates. For example, neutralization of carboxyl groups on P450R by
1-ethyl-3(3-dimethyl-aminodipropyl)carbodiimide (EDC) has been shown to
inhibit both cytochrome c reductase activity and cytochrome
P450-dependent monooxygenase activity, with no effect on electron
transfer to ferricyanide or cytochrome b(27, 28, 29) .
Modification of Lys
of CYP2B4 with fluorscein
isothiocyanate has been shown to inhibit reductase-dependent but not
cumene-hydroperoxide-dependent monooxygenase activity (30) and
Shimizu et al.(31) have identified by site-directed
mutagenesis seven lysyl and/or arginyl residues in CYP1A2 which affect
cytochrome P450-dependent monooxygenase activity. In contrast, studies
on the ionic strength dependence of P450 reduction argue against
charge-pairing between P450R and cytochrome P450 (32, 33, 34) .
In attempts to identify
specific side chain interactions between P450R and substrate, Nisimoto (26) characterized an EDC cross-linked complex between P450R
and cytochrome c, where a lysyl residue in cytochrome c was covalently linked to an acidic residue in the region between
residues 200-220 of the reductase. This region contains two
clusters of acidic amino acids,
Asp-Asp
-Asp
and
Glu
-Glu
-Asp
; one or more of
which could charge-pair with cytochrome c. This study
investigates the role of these residues in substrate binding and
electron transfer by site-directed mutagenesis.
Construction of the P450R expression plasmid, pOR263, and
methods for the expression and purification of the Escherichia
coli-expressed reductase have been described
previously(14) . For site-directed mutagenesis, a 750-base pair BamHI/SacI fragment from pOR263 was cloned into
M13mp19. Mutagenesis was carried out by the method of Zoller and Smith (35) or by using the oligonucleotide-directed mutagenesis kit
obtained from Amersham Corp. The following oligonucleotides were
synthesized at the University of Wisconsin Biotechnology Center: D207N,
5`-CGTCATCATTACCAAG-3`; D208N, 5`-CCCGTCATTATCACCA-3`; D209N,
5`-TTCCCGTTATCATCAC-3`; E213Q, 5`-ATCCTCTTGCAAGTTC-3`; E214Q,
5`-GAAATCCTGTTCCAAG-3`; E215Q, 5`-GATGAAATTCTCTTCC-3`; D207N/D208N,
5`-TTCCCGTCATTATTACCAAG-3`; E213Q/E214Q/D215N,
5`-ATGAAATTCTGTTGCAAGTTC-3`; D207N/D208N/D209N,
5`-TTCCCGTTATTATTACCAAG-3`. Mutant phage were identified by plaque
hybridization and confirmed by sequencing. The mutant BamHI/SacI fragments were cloned into pOR263, and the
mutant expression plasmids were verified by restriction mapping and
sequencing. Mutants with multiple substitutions were prepared using
combinations of the above oligonucleotides. Cytochrome c and
ferricyanide reductase activities and flavin content were determined as
described previously(14) . Assays in potassium phosphate were
carried out in 271 mM potassium phosphate, pH 7.7, giving an
ionic strength of 765 mM. Assays in KCl contained 10 mM potassium phosphate, pH 7.7, and KCl to achieve the desired ionic
strength. Cytochrome c was prepared by
dissolving 15 mg of cytochrome c
in 0.5 ml
of 10 mM potassium phosphate, pH 7.7, adding 1 mg of sodium
dithionite, and passing the reduced cytochrome c over a 5-ml
Sephadex G10 column to remove excess dithionite. Concentrations of
reduced and oxidized cytochrome c were determined by measuring
the absorbance at 550 nm (
= 21 mM) with and
without added dithionite. Cytochrome P450 was purified from
phenobarbital-induced rat liver microsomes as described by Guengerich
and Martin(36) . Assays for benzphetamine N-demethylation contained 0.19 µM P450R,
0.04-0.72 µM cytochrome P450, 20 µg/ml sonicated
dilauroylphosphatidylcholine, 1 mM benzphetamine, 1 mM NADPH, and 54 mM HEPES, pH 7.4. P450R, cytochrome P450,
and lipid were incubated at 37 °C for 5 min in covered microtiter
plates in a volume of 20 µl. Reactions were then initiated by
addition of 180 µl of HEPES buffer containing NADPH and
benzphetamine. After 4 min, reactions were terminated by addition of 50
µl of 30% trichloroacetic acid. After centrifugation to remove
protein, formaldehyde in the supernatants was assayed by the Nash
method, as described by Werringloer(37) .
Fig. 1shows the amino acid sequence of rat P450R
between residues 200 and 221. This region contains two clusters of
acidic residues which have been identified as being cross-linked by EDC
to cytochrome c(26) . Comparison of the mammalian,
yeast, and bacterial reductases identifies invariant
(Asp) and conserved (Glu
) acidic residues,
as well as invariant or conserved residues at positions 204, 216, 219,
220, and 221. Comparison of the rat reductase sequence with that of Desulfovibrio vulgaris flavodoxin reveals identity at
positions 208, 215, and 219 as well as the conservation of an acidic
residue at position 214. The results presented here focus on the
effects of introduction of the corresponding amides for the acidic
residues in these two clusters, changes which produce minimal steric
effects but remove charge-pairing interactions and introduce the
potential for hydrogen-bonding.
Figure 1: Sequence comparison of the region of NADPH-cytochrome P450 oxidoreductase containing the two clusters of acidic amino acids involved in interactions with cytochrome c and cytochrome P450. Residues are identified by the standard single-letter amino acid code. Position numbers are shown at the right of each sequence. Acidic residues 207-209 and 213-215 of the rat enzyme are shown above the sequence. Dots indicate identical residues and dashes(-) indicate gaps introduced to align the sequences. The mammalian sequences are 90% homologous and are represented by the rat sequence. The rat(38) , Saccharomyces cerevisiae(39) , Candida tropicalis(40) , P450-BM3(41) , and Desulfovibrio vulgaris flavodoxin (42) sequences were aligned using the program AALIGN (DNASTAR, Madison, WI).
Table 1shows that
substitution of asparagine for Asp affects primarily the
interaction of P450R with cytochrome P450, as evidenced by a 63%
reduction in the P450-dependent benzphetamine N-demethylase
activity and no effect on cytochrome c and ferricyanide
reduction. Similar changes at either positions 207 or 209 had no effect
on the catalytic activity with cytochrome P450, cytochrome c,
or ferricyanide. The activity of the double mutant D207N/D208N was
similar to that of D208N. The drop in N-demethylase activity
was accompanied by a decrease in k
of 55% for
D208N and 66% for D207N/D208N, with no change in K
(Table 2). No significant
changes in K
or K
were seen in any of the
cluster I single mutants.
In contrast to the results seen with cluster I substitutions, mutations in the second acidic cluster affected interaction of the reductase with cytochrome c but not with cytochrome P450 (Table 3). None of the cluster II substitutions affected P450-dependent N-demethylase activity; however, under standard assay conditions (0.27 M potassium phosphate, pH 7.7), cytochrome c reductase activity of the E213Q mutant was decreased by 59%. E214Q, D215N, D207N/E214Q, and D207N/D215N exhibited normal catalytic activities, while the cytochrome c reductase activities of the D207N/E213Q and E213Q/E214Q/D215N mutants were decreased by 70%. As with cluster I mutations, ferricyanide activity was not affected by any of the cluster II mutations.
Each of the cluster I and cluster II single mutants and mutants carrying two or three substitutions had the expected flavin stoichiometry, i.e. 1 mol of FMN and 1 mol of FAD per mol of protein. Replacement of all six acidic residues with their corresponding amides, however, produced a protein with the normal complement of FAD but only 0.4 mol of FMN/mol of protein. The specific activity of this six-place mutant was 10% of wild type and was significantly less than that of E213Q and E213Q/E214Q/D215N. This reduced activity is probably the result of structural perturbations affecting FMN binding.
Examination of the kinetic properties of the
cluster II mutants in 0.27 M potassium phosphate, pH 7.7,
shows that the decreased specific activity of the E213Q mutant was
accompanied by a 60% decrease in k and a 47%
decrease in K
(Table 4).
Similar decreases were observed in each of the mutants carrying the
E213Q substitution (D207N/E213Q, E213Q/E214Q/D215N, and
D207N/D208N/D209N/E213Q/E214Q/D215N). In contrast, K
was increased 1.6-fold for
the E214Q and D207N/E214Q mutants, with no change in k
. Kinetic properties of the D215N mutant were
the same as wild type. K
and
benzphetamine N-demethylase activity were not affected by any
of the cluster II mutations. Substitution at Glu
produced
a slight, but statistically insignificant, decrease in K
. However, K
for the three- (E213Q/E214Q/D215N) and six-place
(D207N/D208N/D209N/E213Q/E214Q/D215N) mutants was significantly
decreased to 58 and 44%, respectively, of wild type.
The effects of
ionic strength on the catalytic activities of the wild-type and mutant
enzymes are shown in Fig. 2. When ionic strength was increased
by increasing the concentration of potassium phosphate (Fig. 2A), maximum cytochrome c reductase
activity was observed at an ionic strength of approximately 452 mM for the wild-type enzyme, 339 mM for the E213Q enzyme,
and 678 mM for the E214Q enzyme. Maximum activity of the
E213Q/E214Q/D215N mutant was observed at 502 mM (not shown).
When ionic strength was varied by increasing the concentration of KCl (Fig. 2B), wild-type k increased
to a maximum in the range between 208 and 479 mM, while E213Q k
was maximal at 208 mM and declined
thereafter. k
for the E214Q enzyme was maximal
at ionic strengths greater than 479 mM. While k
of the E213Q enzyme was only 28% of wild type
at 749 mM, it was the same as that of wild type at low ionic
strength.
Figure 2:
Effect
of ionic strength on cytochrome c reductase activity. A, dependence of cytochrome c reductase activity on
ionic strength. Cytochrome c reductase activities were
measured in potassium phosphate, pH 7.7, at the indicated ionic
strength. (), wild type; (
), E213Q; (
), E214Q. Values
are the average of two separate experiments. B, dependence of k
for cytochrome c reduction on ionic
strength. Cytochrome c reductase activities were measured in
10 mM potassium phosphate, pH 7.7, with varying amounts of
KCl. (
) wild type; (
) E213Q; (
) E214Q. Values are
mean ± S.D. for three separate
experiments.
The ionic strength dependence of K for the wild-type, E213Q, and E214Q proteins is shown
in Table 5. For all three proteins, K
increased with increasing ionic strength. At each
ionic strength, K
for E213Q
was lower than that found for either the wild-type or E214Q enzymes;
this difference was more pronounced at higher ionic strengths. In
addition, K
for the wild-type
enzyme was also dependent upon the anion, being 34.6 µM at
749 mM ionic strength with KCl (Table 5) and 21.1
µM at 765 mM ionic strength using potassium
phosphate (Table 4). This anion dependence was not observed with
E213Q and E214Q ( Table 4and Table 5).
Cytochrome c was a competitive inhibitor versus cytochrome c
. Cytochrome c
K
for the
wild-type enzyme was dependent upon ionic strength, with K
at 118 mM being 7 times
greater than that at high (749 mM) ionic strength (Table 5). A similar trend was noted for the E214Q mutant;
however, cytochrome c
K
for the E213Q mutant was similar to wild type at high ionic
strength but did not increase with decreasing ionic strength.
The
ionic strength dependence of many electron transfer reactions has been
proposed to be a consequence of complementary electrostatic
interactions necessary for formation and/or stabilization of productive
electron transfer
complexes(19, 20, 21, 43) . This
ionic strength dependence is also observed for the reaction of P450R
with cytochrome c. Fig. 3shows that k/K
for
the wild-type enzyme decreases as ionic strength increases. The E214Q k
/K
shows a similar ionic strength dependence, but is shifted to lower
ionic strengths, consistent with removal of a weak electrostatic
interaction. The E213Q mutant displays a different ionic strength
dependence, probably because this substitution alters interactions with
both oxidized and reduced cytochrome c.
Figure 3:
Dependence of
k/K
on ionic
strength. Cytochrome c reductase activities were measured in
10 mM potassium phosphate, pH 7.7, with varying amounts of
KCl. Actual k
and K
values used to calculate k
/K
are
found in Fig. 2B and Table 5(
), wild-type;
(
), E213Q; and (+), E214Q. The solid lines are fits
of the wild-type and E214Q data to the Watkins et al.(45) model using the monopole term only. The dotted
line is a fit of the E213Q data to the Watkins model (monopole
only) and the dashed line is a fit with an additional dipolar
term.
A number of theoretical models have been developed to describe the effects of ionic strength on biomolecular rate constants when electrostatic interactions are important in formation of the electron transfer complex. Models such as that of Wherland and Gray (44) treat the interacting species as small and uniformly reactive, while Watkins et al.(45) have developed a model incorporating asymmetric protein surface charge distributions and localization of charges at the site of electron transfer.
Nonlinear least-squares fits of the ionic
strength dependence of k/K
to
the Watkins model are shown in Fig. 3for the wild-type, E213Q,
and E214Q proteins. The wild-type and E214Q proteins fit the model
quite well, yielding 5.8 and 20.2 Å, respectively, for the radius
of the interaction domain. These values compare favorably with radii of
7-20 Å obtained for other electrostatically stabilized
electron transfer complexes(44) . Similar fits and radii for
these two proteins were obtained with the Wherland-Gray model (not
shown). These results are consistent with the presence of electrostatic
interactions in the transition state which are weakened by removal of
Glu
. The ionic strength dependence for E213Q, however,
did not fit these simple electrostatic models, giving poor fits to both
the Watkins and Wherland-Gray equations. Addition of dipole terms to
the Watkins model improved the fit (Fig. 3); however, the
interaction radius remained unreasonably small (1.4 Å).
The
kinetic constants shown in Table 5and Fig. 2and Fig. 3can be used to obtain the changes in the free energies of
binding for wild-type, E213Q, and E214Q reaction intermediates when
ionic strength is varied from 118 to 749
mM(46, 47) . Fig. 4shows the effects
of ionic strength on the relative free energies of binding for the
reductase-cytochrome c ground state,
reductase-cytochrome c transition state, and
reductase-cytochrome c
complexes of the
three enzymes, as well as on the activation energy. For all three
enzymes, increasing the ionic strength from 118 to 749 mM increased the free energies of binding of both the ground state
and transition state complexes. For the wild-type and E214Q proteins,
the ground state complexes were destabilized more than the transition
state, producing, respectively, 209 and 354 cal/mol decreases in the
activation energies and leading to the observed increases in k
at high ionic strength. The opposite case
holds for the E213Q enzyme, where destabilization of the ground state
complex was less than that of the transition state by 509 cal/mol,
accounting for the observed decrease in k
at
high ionic strength.
Figure 4:
Effect
of increasing ionic strength from 118 to 749 mM on relative
free energies of binding of wild-type (), E213Q (
), and
E214Q (&cjs2112;) reductase-cytochrome c reaction
intermediates and activation energies. Values shown are changes in the
relative free energies of binding of P450R-cyt c
, the reductase-cytochrome c
ground state complex (-RT ln(K
/K
));
P450R-cyt c
, the reductase cytochrome c transition state complex (-RT ln((k
/K
)
/(k
/K
)
));
activation energy, (-RT ln(k
)
/(k
)
);
and P450R-cyt c
, the reductase-cytochrome c
complex (-RT ln(K
/K
)).
Increasing the ionic strength from 118 to 749
mM stabilized the wild-type and E214Q reductase-cytochrome c complexes by nearly 1 kcal/mol but had no
effect on the E213Q reductase-cytochrome c
complex (Fig. 4). At low ionic strength, the E213Q
reductase-cytochrome c
complex was
stabilized by 1.2 kcal/mol relative to wild type. At high ionic
strength, however, this substitution had no effect on binding of
cytochrome c
.
The current study examines the role of two clusters of acidic
amino acids, located in the amino-terminal half of P450R, in substrate
recognition. The results indicate that the sites and mechanisms of
interaction are distinct for the two substrates, as well as for the
oxidized and reduced forms of cytochrome c. Asp of cluster I affects P-450 catalyzed N-demethylation
while Glu
of cluster II influences cytochrome c reduction. Although this region is adjacent to the FMN binding
domain(8, 12, 13, 14) , none of
these substitutions, with the exception of the
D207N/D208N/D209N/E213Q/E214Q/D215N substitution, affected FMN content.
Ferricyanide reductase activity was also unchanged, suggesting that the
observed effects are not due to conformational changes in the vicinity
of the FAD/NADPH domain.
Only Asp located in cluster I
interacts measurably with cytochrome P450. Importantly, mutagenesis of
adjacent residues (Glu
and Glu
) did not
alter any enzymatic properties. Furthermore, activity of the double
mutant, D207N/D208N, was similar to that of D208N, suggesting a
specific interaction with Asp
. Since K
was unchanged and k
and k
/K
were reduced 54 and 56%, respectively, Asp
appears to be
involved in interactions affecting electron transfer rather than
cytochrome P450 binding, either directly or indirectly by orienting the
P450R cytochrome P450 complex for optimum electron
transfer(48, 49) .
In contrast, interaction of
P450R with cytochrome c involves primarily the second acidic
cluster (residues 213-215). The largest changes in cytochrome c reductase activity were seen with the E213Q mutant, where
catalytic activity was reduced 59% and Kwas reduced 47%. Conversion of Asp
to its
corresponding amide affected only the ionic strength dependence of k
and K
. The D215N substitution had no effect on catalytic
activity. Properties of the double and triple mutants were similar to
those of the corresponding single mutants, suggesting that each residue
interacts independently with cytochrome c.
The ionic
strength dependence of wild-type and E214Q k/K
values fits well for models incorporating electrostatic
interactions and is consistent with removal of a weak electrostatic
interaction in the E214Q mutant (Fig. 3). In contrast, the ionic
strength dependence of E213Q does not fit these models without the
introduction of additional terms, either as a result of an unfavorable
conformation of the electron transfer complex or as a consequence of
altered interactions with reduced cytochrome c.
Comparison
of the effects of ionic strength on the kinetic properties of the
wild-type, E213Q, and E214Q enzymes permits the identification of the
reaction intermediates affected by changes in salt concentration and by
removal of either the Glu or Glu
side
chains (Fig. 4). For the wild-type enzyme, increasing ionic
strength increased the free energy of binding for both the ground state
and transition state complexes, consistent with the presence of
electrostatic interactions between P450R and cytochrome c.
High ionic strength increased the binding energy of the ground state
complex more than that of the transition state, leading to a net
decrease in activation energy and the observed increase in k
. This is an example of the use of the free
energy of binding of the enzyme-substrate complex to increase the rate
of catalysis(46) . It is likely that this mechanism also
underlies the inhibition of cytochrome c reduction by polyols
observed by Voznesensky and Schenkman(32, 33) .
Similarly, removal of a weak interaction between Glu
and
cytochrome c has a larger destabilizing effect on the
P450R-cytochrome c ground state complex than on the transition
state complex, leading to a net increase in k
at
high ionic strength.
Replacement of Glu with glutamine
had no effect at low ionic strength; however, at higher ionic
strengths, the ground state complex was more stable than that of the
wild-type enzyme, leading to a net increase in activation energy and
decrease in k
. Glu
in the
wild-type enzyme may interact weakly or not at all with cytochrome c; however, glutamine at position 213 must be able to form
hydrogen bonds with cytochrome c which act to stabilize the
P450R-cytochrome c
complex in a conformation
which is less favorable for electron transfer.
Although Glu does not interact strongly with oxidized cytochrome c,
cytochrome c
K
of the wild-type enzyme decreased with increasing ionic
strength, consistent with the presence of an electrostatic repulsion
between P450R and cytochrome c
(Table 5). The E213Q mutant bound cytochrome c
tightly at both high and low ionic
strength as a result of removal of this repulsive interaction. Based on K
values (Table 5), the E213Q-cytochrome c
complex was stabilized by 965 cal/mol relative to wild type at 118
mM ionic strength, close to the theoretical 1 kcal/mol
destabilization of the flavodoxin-cytochrome c complex
resulting from loss of a positive charge upon reduction of cytochrome c(50) .
The free energy changes associated with the
D208N, E213Q, and E214Q substitutions range from nil to 1.3 kcal/mol,
substantially less than those commonly associated with loss of an ionic
bond. This may be because these substitutions do not affect
rate-limiting steps in benzphetamine demethylation or cytochrome c reduction or because an electrostatic interaction has been
replaced with a hydrogen bonding interaction(47) , as has been
proposed for binding of human adrenal ferredoxin to
P450(51) . Free energy changes of this magnitude
are also to be expected if multiple electrostatic, hydrophobic, and van
der Waals interactions contribute to binding. Mutagenesis of cationic
residues of P450
(23) and cytochrome b
(24) which are involved in
charge-pairing interactions with putidaredoxin and cytochrome b
reductase, respectively, produced binding energy
changes similar to those observed here, and it was proposed that a
single electrostatic interaction made a relatively small contribution
to overall binding. The altered ionic strength dependence of the E213Q
mutant may be an indication of an increased contribution from these
other interactions.
In summary, these results demonstrate that
residues located in two acidic clusters identified by chemical
cross-linking experiments have specific effects on the interaction of
P450R with cytochrome c and cytochrome P450. Cytochrome c and cytochrome P450 interact with separate residues in this
region. Asp of P450R influences electron transfer to
cytochrome P450 with no effect on binding. Although additional factors
are necessary for electron transfer to cytochrome
P450(31, 32, 33, 34, 52) ,
the ionic strength data indicate that electrostatic interactions do
predominate in formation of the P450R-cytochrome c complex (Fig. 3)(6, 52) . Glu
interacts
weakly with cytochrome c and Glu
is involved in
a repulsive interaction with reduced cytochrome c. Glu
may not form contacts with oxidized cytochrome c;
however, replacement of Glu
with glutamine may introduce
new hydrogen bonding interactions which stabilize the
reductase-cytochrome c complex in an altered conformation
where electron transfer is less efficient. The effects of ionic
strength on the kinetic properties of these mutants point toward
multiple charge-pairing, hydrophobic, and repulsive interactions
involving these residues and other, as yet unidentified residues, which
must also play a role in electron transfer from P450R to its
substrates.