(Received for publication, August 9, 1996, and in revised form, November 6, 1996)
From the Department of Medical Biochemistry, Ohio
State University, Columbus, Ohio 43210 and § Phytotechnology
Research Center and Department of Biological Sciences, Michigan
Technological University, Houghton, Michigan 49931
The recombinant NADH-cytochrome c
reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1),
consisting of the contiguous heme-containing cytochrome b
domain and flavin-containing NADH-cytochrome b reductase
fragment, has been characterized spectroscopically and kinetically.
Reductive titration with sodium dithionite indicates heme reduction
takes place prior to flavin reduction, which correlates well with the
reduction potentials for enzyme-bound heme (15 mV) and FAD (280 mV).
Reductive titration with NADH also indicates that the reduced enzyme
forms a charge-transfer complex with NAD+. The circular
dichroism spectrum of the oxidized fragment is primarily due to the
flavin, whereas the ferrous heme dominates the circular dichroism
spectrum of reduced enzyme. Three kinetic phases are observed in the
course of the reaction of the enzyme with NADH, each with a distinct
spectral signature. The fast phase represents flavin reduction,
concomitant with the formation of a charge-transfer complex between
reduced flavin and NAD+, and exhibits hyperbolic dependence
on NADH concentration with a Kd of 3 µM and a limiting rate constant of 560 s
1.
Electron transfer from reduced flavin to heme with a rate constant of
12 s
1 is the intermediate phase, which is rate-limited by
breakdown of the charge-transfer complex between NAD+ and
reduced flavin. The slow phase is dismutation of a pair of molecules of
two-electron reduced enzyme (generated at the end of the second phase
of the reaction) to give one molecule each of one- and three- electron
reduced enzyme, with a second order rate constant of 2 × 106 M
1 s
1. In the
presence of excess NADH, this dismutation reaction is followed by the
rapid reaction of the one-electron reduced enzyme with a second
equivalent of NADH to generate fully reduced enzyme. On the basis of
this work, it appears that dissociation of NAD+ from the
reduced flavin site rate limits electron transfer to the cytochrome and
likely represents the overall rate-limiting step of catalysis.
Nitrate reductase (EC 1.6.6.1-3) catalyzes the pyridine nucleotide-dependent reduction of nitrate to nitrite as the first and rate-limiting step in nitrogen assimilation in algae, fungi, and higher plants (1-3). The enzyme is a complex metalloflavoenzyme of molecular weight 200 kDa containing an FAD, a b-type cytochrome, and a molybdopterin center in each of its two identical subunits (1-3). In the course of the catalytic sequence, reducing equivalents (in the form of NADH) enter the enzyme at the flavin site and are subsequently transferred via the heme to the molybdenum center, where the chemistry associated with nitrate reduction takes place. The redox-active centers are present in structurally independent domains, which are laid out in a linear fashion in the amino acid sequence and which exhibit homology to other enzymes containing similar cofactors (2-4). The N-terminal molybdenum-containing fragment of nitrate reductase possesses sequence homology to the molybdenum binding region of sulfite oxidase (5, 6), the central heme domain to the soluble domain of mammalian and higher plant cytochrome b5 (3) and the C-terminal fragment of nitrate reductase (containing only the FAD) to the physiological partner of cytochrome b5, namely, NADH-cytochrome b5 reductase, as well as other members of the ferredoxin-NADP+ reductase family (2-4). The FAD-containing recombinant cytochrome b reductase fragment of corn leaf nitrate reductase has been characterized recently, and the role of cysteine 242 (invariant among nitrate reductases) examined (7, 8). An x-ray crystal structure for this fragment is now available and has established that the protein is a member of the structurally related but functionally diverse group of flavin reductases that comprise the ferredoxin-NADP+ reductase family (9).
The central heme domain is connected to the N- and C-terminal domains through hinge regions that are susceptible to proteolytic cleavage. Native nitrate reductase can be easily cleaved into stable fragments that retain partial catalytic activities characteristic of their prosthetic groups (10). The structural independence of the cofactor-binding domains of nitrate reductase is further supported by the ability to independently express the FAD- and heme-containing fragments as stable and redox-active polypeptides (11-13). The recombinant FAD-containing cytochrome b reductase fragment has NADH-ferricyanide activity and an absorbance spectrum similar to mammalian cytochrome b5 reductase (11). The heme domain, when reduced by sodium dithionite, is able to pass its electron on to cytochrome c (12), and when the heme domain and cytochrome b reductase fragment are expressed as a single polypeptide, the protein exhibits NADH-cytochrome c reductase activity (13). The absorbance spectrum of this cytochrome c reductase fragment of nitrate reductase is virtually indistinguishable from that of native nitrate reductase, with the cytochrome b dominating the spectrum of both oxidized and reduced enzyme. Although a three-dimensional structure for this fragment is as yet unavailable, docking studies have been performed using the structures for the cytochrome b reductase fragment and bovine cytochrome b5, which exhibits 47% amino acid residue identity and 84% sequence homology with the cytochrome b domain of nitrate reductase (14). The resulting model suggests that the propionate group on the heme comes in close proximity to the C-8 methyl group of the FAD in order for electron transfer from FAD to heme to occur, in a manner similar to flavocytochrome b2 (15).
Here we report the characterization of the spectroscopic and kinetic properties of the recombinant cytochrome c reductase fragment of spinach nitrate reductase. Our results indicate that in the course of turnover, electron transfer from the flavin to the heme does not occur until NAD+ has dissociated from the flavin and that this is likely to be the rate-limiting step of catalysis.
Spinach leaf NADH-nitrate reductase cDNA clone pSPNR117, which contains a 2.3-kilobase insert, was used to prepare the fragment coding for spinach CcR1 (16; GenBankTM Accession No. U08029[GenBank]). The pSpNR117 clone was partially digested with XhoII to obtain a 1.6-kilobase fragment which was subcloned into Bluescript at the BamHI site. When this subclone was digested with EcoRI, the fragment obtained was subcloned into the unique EcoRI restriction site of the Pichia expression vector pHIL-D2, which is a commercially available vector designed for intracellular expression of proteins (Invitrogen). This construct was used for CcR expression in Pichia pastoris strain his4 GS115, per the instruction manual provided by the manufacturer. The CcR containing pHIL-D2 plasmid was linearized by NotI digestion and transformed into Pichia using the spheroplast transformation method (17). A transformant with slow growth on methanol was selected, which indicated that the CcR coding fragment was most likely integrated in the aox1 gene locus of Pichia. Thus, the CcR expressing transformant is of the type called mutS, for slow methanol utilization, since the alcohol oxidase I of Pichia is knocked out and only alcohol oxidase II remains active.
The CcR expressing Pichia transformant was grown at 30 °C in 50 ml of BMGY medium for 2 days and then used to inoculate 1 liter of BMGY medium in a 2.4-liter flask, which was grown in a shaking incubator (280 rpm) at 30 °C. After 2 days of incubation, the cells were harvested by centrifugation and resuspended in 1 liter of BMM medium. For CcR induction, the cells were grown with the addition of 10 ml of 100% methanol every 24 h. After 5 days of growth in methanol-containing medium, the cells were harvested by centrifugation and resuspended in 500 ml of phosphate extraction buffer (50 mM NaPO4, pH 7.3, 1 mM EDTA). Cells were disrupted using glass beads in a Bead-Beater extraction device (Biospec Products). The extract was centrifuged (18,000 × g, 20 min at 4 °C), and a 30-50% ammonium sulfate precipitate was prepared which was suspended in phosphate extraction buffer. The ammonium sulfate fraction was applied to blue Sepharose, and after washing with phosphate extraction buffer, the CcR was eluted with NADH as described previously (11). Residual NADH was removed and the buffer exchanged to 25 mM MOPS, pH 7.0, 1 mM EDTA using a DIAFLO ultrafiltration stirred cell (Amicon). Purity of the CcR preparations was determined by denaturing polyacrylamide gel electrophoresis as described previously (7).
Protein sequencing of the purified CcR revealed that the second available AUG in the spinach nitrate reductase coding sequence in the Pichia vector construct was where translation had begun. Thus, the CcR fragment expressed here contains spinach nitrate reductase residues 253-926 with a predicted Mr = 41,475. Careful size analysis of the denaturing polyacrylamide gel of purified CcR indicated that the recombinant protein is 41.7 kDa, which confirmed the predicted size. More details of the preparation and properties of the Pichia expressed CcR will be published elsewhere.
All experiments with the recombinant CcR were performed in 0.1 M MOPS, 0.1 N KCl, pH 7.0, containing 0.1 mM EDTA, unless otherwise stated. Ultraviolet/visible spectroscopy was carried out using a Hewlett-Packard 8452A diode array spectrophotometer, and circular dichroism (CD) spectroscopy was carried out on an AVIV 40DS spectrophotopolarimeter. Rapid mixing experiments were performed using a Kinetic Instruments Inc. stopped-flow apparatus interfaced to an On-Line Instrument Systems (OLIS) model 3920Z data collection system. Enzyme samples, in a tonometer equipped with a three-way stopcock valve with a male Luer connector, were made anaerobic by alternately evacuating and flushing with oxygen-free argon on an anaerobic train. The tonometer was mounted on the stopped-flow apparatus and the enzyme rapidly mixed with anaerobic solutions of NADH. Kinetic transients obtained were analyzed and the rate constants determined using OLIS software.
EPR spectra were recorded at 150 K using a Brüker Instruments ER 300 spectrometer equipped with a ER 035 M gaussmeter and a Hewlett-Packard 5352B microwave frequency counter. EPR samples were collected using a rapid quench apparatus that permitted the collection of liquid samples directly into EPR tubes, which were then frozen immediately using a dry ice/acetone bath.
Reductive titrations were carried out in anaerobic glassware described
previously (18). Samples were made anaerobic and then titrated with
solutions of either sodium dithionite or NADH in 0.1 M
MOPS, 0.1 N KCl, pH 7.0, containing 0.1 mM EDTA
that had previously been made anaerobic by bubbling with oxygen-free argon. The reduction potential of the cytochrome b cofactor
was determined as described by Massey (19) using
5-hydroxy-1,4-naphthoquinone (Eo
=
3 mV)
(20).
NADH and 5-hydroxy-1,4-naphthoquinone were obtained from Sigma, and sodium dithionite was obtained from Virginia Chemical Co. All other chemicals were of reagent grade and used without additional purification.
The visible absorption spectrum (Fig.
1A) of the oxidized cytochrome c
reductase fragment of spinach nitrate reductase is virtually identical
to that of the holoenzyme from a variety of sources. The predominant
contribution to the spectrum is that of the cytochrome b
cofactor with the strongest absorption peak being the Soret band at 414 nm and two broad bands in the region between 500 and 600 nm; the FAD of
the protein contributes principally at ~460 nm. When the enzyme is
reduced with sodium dithionite, the Soret band shifts to 424 nm and new
bands appear at 528 nm and 556 nm; there is a loss of the shoulder at
480 nm due to the bleaching of the FAD upon reduction.
The CD spectrum (Fig. 1B, broken line) of oxidized cytochrome c reductase is mainly due to the flavin and is very similar to that observed for the corn cytochrome b reductase fragment (8). The contribution from the heme domain is primarily in the region between 550 and 600 nm as a small negative band. On the other hand, the CD spectrum of the reduced enzyme (Fig. 1B, solid line) is dominated by the ferrous heme, and the contribution from the flavin hydroquinone, which absorbs weakly above 400 nm, is negligible. The CD spectrum of the reduced enzyme is similar to that observed for the heme domain of the enzyme from Chlorella vulgaris obtained by limited proteolysis (21).
Reductive and Potentiometric TitrationsEquilibrium reductive
titrations with dithionite (Fig. 2A) and NADH
(Fig. 2B) have been carried out. At pH 7.0, the cytochrome b is reduced earlier in the course of the titration than is
FAD, as is seen by a plot of the fractional absorption change at 460 nm
(predominantly due to reduction of the flavin) versus 556 nm (due to reduction of the heme) (Fig. 2A, inset). This
behavior is expected in light of the significantly higher reduction
potential of the heme relative to the flavin (see below). Similarly,
with NADH as the reductant, heme reduction occurs earlier in the course of the titration than does FAD reduction (despite it being known that
the flavin site is the site of initial reduction by NADH). During the
titration with NADH, concomitant with the eventual onset of flavin
reduction, there is a net increase in long wavelength absorption (Fig.
2B, inset) indicating the formation of a charge-transfer complex between reduced flavin and NAD+ comparable to that
observed with the cytochrome b reductase fragment of nitrate
reductase (8).2 In the course of this
equilibrium titration, there is no evidence for accumulation of either
anionic or neutral flavin semiquinone. Since NADH is an obligatory
two-electron donor, this observation indicates not only that the
semiquinone is thermodynamically destabilized (i.e. that the
quinone/semiquinone couple is lower than the semiquinone/hydroquinone couple) but that dismutation of the two-electron reduced enzyme initially generated by reaction of NADH with the flavin center (possessing reduced heme and flavin semiquinone) is rapid.
The potential of the cytochrome box/cytochrome
bred couple has been determined by the method
described by Massey (19) in which a reductive titration of an unknown
(a redox center in a protein) is carried out in the presence of an
equivalent amount of a standard (a redox dye for which the reduction
potential is known). A plot of the logarithm of oxidized/reduced
concentrations of the unknown versus the logarithm of the
oxidized/reduced concentration of the standard dye (22), in this case,
5-hydroxy-1,4-naphthoquinone (Eo
=
3 mV)
(Fig. 3) yields a reduction potential for the cytochrome b of 15 mV. This value is comparable to that reported by
Cannons et al. (12) for the recombinant cytochrome
b domain of nitrate reductase from C. vulgaris
but is considerably higher than that reported for cytochrome
b in holo-nitrate reductases from different sources (23,
24). In the enzyme from C. vulgaris this difference has been
attributed to a modulation of the heme reduction potential by the
presence of a portion of the molybdopterin-binding region polypeptide
(12).
Reaction of Cytochrome c Reductase with Excess NADH
The
reaction of oxidized cytochrome c reductase fragment with a
pseudo-first-order excess of NADH (that is, under conditions where
[NADH] [CcR]) has been examined by rapidly mixing the enzyme
with NADH under anaerobic conditions at 10 °C (it being found that
lower temperatures are required to slow the reaction sufficiently to be
observed in the stopped-flow instrument). Reduction of flavin in these
experiments can be monitored conveniently at 460 nm, reduction of the
cytochrome b monitored at 556 or 424 nm, and formation of
the charge-transfer complex (FADH2·NAD+)
monitored at 750 nm. Three kinetic phases are observed in the course of
this reaction (Scheme 1). The fast phase, due to the reduction of the flavin, is complete in ~10 ms and is observed as a
decrease in absorbance at 460 nm (Fig. 4A).
Concomitant with the decrease in absorbance at 460 nm, an increase in
absorbance is observed at 750 nm, indicating formation of the
charge-transfer complex between the reduced flavin and NAD+
at the same rate as reduction of flavin. The fast phase of the reaction
is found to exhibit hyperbolic dependence on the concentration of NADH,
and from a double reciprocal plot a Kd of 3 µM and a kreduction of 560 s
1 are obtained (Fig. 4A, inset). These values
are in agreement with previous studies of the reaction of the corn
cytochrome b reductase fragment with NADH (Table
I) (8), indicating that the initial reduction of the
flavin by NADH is largely independent of the presence of the heme
domain.
Scheme 1.
|
The spectral change associated with the intermediate phase, with a
large absorbance change in the Soret and an absorbance increase at 556 nm, indicates that the process involves the transfer of one electron
from the flavin to the cytochrome b (Fig. 4B), with a rate constant of 12 s1; this rate constant is
independent of NADH concentration. Breakdown of the charge-transfer
complex, as reflected in loss of long wavelength absorbance,
accompanies heme reduction, suggesting strongly that electron transfer
from the flavin to the cytochrome b is limited by the
breakdown of the charge-transfer complex formed between reduced flavin
and NAD+ (presumably via product dissociation; see below).
The slow phase of the reaction is manifested as a subsequent increase
in absorbance at both 556 nm (Fig. 4B) and 750 nm (Fig.
5A) and reflects further heme reduction and
accumulation of the charge-transfer complex. The most likely mechanism
whereby this occurs is via a dismutation of the flavin semiquinone
(remaining after transfer of one electron to the heme) to give a
mixture of fully oxidized and fully reduced flavin, followed by the
reaction of the former with a second equivalent of NADH to regenerate
the charge-transfer complex. The dismutation of the flavin semiquinone
is not described by a single exponential, but rather is a
strictly second-order reaction with a rate constant of 2.11 × 106 M
1 s
1,
indicating that the dismutation is a bimolecular
process.3
Reaction of Cytochrome c Reductase with One Equivalent of NADH
Reaction of the cytochrome c reductase fragment with one equivalent of NADH (approximately 14 µM each) is again triphasic. The absorbance change associated with the fast phase is essentially identical to that seen in the reaction with excess NADH, with an absorbance decrease at 460 nm and an increase in absorbance at 750 nm, indicating flavin reduction and simultaneous formation of the charge-transfer complex (data not shown). The intermediate kinetic phase representing the one electron reduction of cytochrome b is also observed, again as an increase in absorbance at 556 nm and an absorbance decrease at 750 nm. The absorbance associated with the slow phase of the reaction under conditions of stoichiometric NADH, however, is quite distinct from that observed in the presence of excess NADH (Fig. 5B). Under conditions of excess NADH, dismutation is followed by re-reduction of the 50% of the protein containing oxidized flavin and quantitative accumulation of the charge-transfer complex. With stoichiometric NADH, this re-reduction cannot take place and the dismutation reaction leaves a 1:1 mixture of enzyme possessing fully oxidized and fully reduced flavin (each with the heme center reduced). The former species cannot form the charge-complex because the flavin is in the incorrect oxidation state. The latter species can (and does) form a certain amount of charge-transfer complex, as reflected in the transient shown in Fig. 5B, which does not return to zero absorbance at the end of the slow phase. The incomplete formation of charge-transfer complex with the fully reduced enzyme is presumably due to the relatively low concentration of NAD+ at the conclusion of the reaction. It is to be noted that to the extent the dismutation reaction is incomplete, the two-electron reduced enzyme that remains will also be unable to form the charge-transfer complex, as the electron distribution within the protein favors reduced heme and flavin semiquinone over oxidized heme and flavin hydroquinone (as reflected in the large spectral change associated with the intermediate phase of the reaction with NADH whose wavelength dependence indicates that the process reflects heme reduction at the expense of the initially formed flavin hydroquinone). Again the flavin is in the incorrect oxidation state for formation of the charge-transfer complex. The principal factor which determines whether the charge-transfer complex is formed in the experiments with excess or stoichiometric [NADH] is its kinetic (as opposed to thermodynamic) stability.
In order to examine the presumed flavin semiquinone species generated
upon electron transfer from the flavin hydroquinone to the heme
(i.e. that formed at completion of the second phase of the
reaction), a quench experiment was performed, aimed at stopping the
reaction of the cytochrome c reductase fragment with NADH
prior to dismutation of the flavin semiquinone. The cytochrome c reductase fragment was rapidly mixed with one equivalent
of NADH in a rapid quench apparatus, and at 4 °C and samples were collected directly into EPR tubes and frozen immediately in a dry
ice/acetone bath. The EPR spectrum of the flavin semiquinone (Fig.
6) yielded a signal with a line width of approximately
13 G, indicating the accumulation of the anionic form of flavin
semiquinone.
The visible absorption spectrum of the recombinant cytochrome
c reductase fragment of spinach nitrate reductase is, as
expected, dominated by the cytochrome b cofactor and is
virtually indistinguishable from that reported for the native enzyme
(2). This indicates that the contribution from the molybdenum domain to
the absorption spectrum of native enzyme is extremely small. This
observation is consistent with the results reported for the enzyme
sulfite oxidase (26), whose molybdenum domain exhibits high degree of sequence homology with that of nitrate reductase. The molybdenum domain
of sulfite oxidase exhibits two broad bands at around 350 nm and
between 450 and 500 nm, with an extinction coefficient of about 1600 M1 cm
1 at 480 nm. The CD
spectra of oxidized and reduced CcR reported here are also consistent
with those reported for the cytochrome b reductase fragment
of corn nitrate reductase (8) and the flavin and heme domains of
nitrate reductase from C. vulgaris (12). The CD spectrum of
the oxidized enzyme is predominantly due to the flavin domain, whereas
that of the reduced enzyme is principally due to the cytochrome
b. Particularly in the case of the flavin domain, the close
similarity of the flavin contribution to the CD of oxidized the
flavin-containing cytochrome b reductase fragment of nitrate
reductase (8), CcR (present work), and native nitrate reductase (12)
suggests that there is little perturbation of the flavin environment in
going from one protein form to another.
The reduction potential determined for the cytochrome b of
the cytochrome c reductase fragment (E = 15 mV) (Table I) is considerably higher than that reported for native
nitrate reductase (
E = -123 mV) (23) but corresponds
well to the value reported by Cannons et al. for the heme
domain of the nitrate reductase from C. vulgaris
(
E = 16 mV) (12). Cannons et al. (12)
have demonstrated that while the cytochrome b domain in
isolation has a rather high potential, increasing the size of the
cytochrome b domain to include a part of the molybdenum
binding portion of the protein results in a decrease in the potential
of the cytochrome b by 44 mV, suggesting that residues in
the molybdenum domain are involved in modulating the cytochrome
b reduction potential. Our results indicate that there is no
comparable effect seen with the flavin domain and that the cytochrome
b reduction potential in the cytochrome c
reductase fragment is similar to that of the isolated cytochrome
b. The heme potential being so much higher than the flavin
potential (
E =
280 mV) (23) is consistent with the
observation that during equilibrium reductive titrations with either
sodium dithionite or NADH the heme is reduced prior to the flavin
(present work).
The reaction of the cytochrome c reductase fragment of
nitrate reductase with NADH is found to be triphasic (Scheme 1), with the three phases representing flavin reduction (fast phase), electron transfer from reduced flavin to the heme concomitant with breakdown of
the FADH2·NAD+ charge-transfer complex
(intermediate phase), and dismutation of the two-electron reduced
enzyme to yield a mix of one- and three-electron reduced enzyme (slow
phase, followed by reaction of the one-electron reduced enzyme with a
second equivalent of NADH when it is present in excess). Only the fast
phase of the reaction is dependent on NADH concentration, with a
hyperbolic dependence reflecting a Kd of 3 µM and a kreduction of 560 s1. Concomitant with reduction of the flavin, a
charge-transfer complex is formed between reduced flavin and
NAD+, as is typical of the ferredoxin-NADP+
reductase family of flavoproteins (27-29). The kinetically determined dissociation constant for NADH and the rate of reduction for the fast
phase of the reaction compare well with the corresponding values
determined for the cytochrome b reductase fragment of corn nitrate reductase (Table I) and indicate that the presence of the heme
domain has little effect on the reaction of NADH with the flavin. After
flavin reduction, electron transfer to cytochrome b takes
place at a relatively slow rate (12 s
1) and is apparently
rate-limited by the breakdown of the charge-transfer complex. Since
electron transfer from the flavin to the heme is highly favorable in
the cytochrome c reductase fragment even in the presence of
NAD+ (the difference in potentials is ~300 mV), this
effect is undoubtedly kinetic rather than thermodynamic in nature. Even
so, under conditions of even a moderate excess of NADH, the relative
rate constants associated with formation and decay of the
charge-transfer complex are such that essentially 100% of the protein
transiently accumulates as the long wavelength-absorbing species.
Subsequent to heme reduction the flavin semiquinone that is left
dismutates to give a 1:1 mixture of cytochrome c reductase fragment possessing reduced heme plus either fully oxidized or fully
reduced flavin. In the presence of excess NADH, the former species is very rapidly reduced by a second equivalent of NADH resulting in 100% formation of the
FADH2·NAD+ charge-transfer complex
(observed as an increase in absorbance at 750 nm at longer reaction
times; Fig. 5A). In the reaction of the cytochrome
c reductase fragment with a stoichiometric amount of NADH,
this reaction is not observed (Fig. 5B), and the slow phase
is instead represented simply by the relatively small change in
absorbance due to the dismutation of the flavin semiquinone. The
thermodynamic favorability of this dismutation indicates that the
reduction potential for the FAD/FAD· couple is
substantially lower than that of FAD·
/FADH2
couple and is consistent with the fact that during a reductive titration of the cytochrome b reductase fragment of nitrate
reductase (which contains only the flavin cofactor) with sodium
dithionite, formation of the flavin semiquinone does not accumulate to
an appreciable extent (8). Dismutation of the flavin semiquinone is
also seen in related flavoenzymes such as cytochrome
b5 reductase (30, 31) and adrenodoxin reductase
(32). In the case of cytochrome b5 reductase,
dismutation occurs only in the absence of the product NAD+,
with the semiquinone species being stabilized in the presence of
product (but not as a charge-transfer complex). The behavior of the
cytochrome c reductase fragment of spinach nitrate reductase is similar to that of adrenodoxin reductase in that it does not stabilize the flavin semiquinone even in the presence of
NAD+, and electron transfer from the reduced flavin to
cytochrome b does not take place prior to breakdown of the
enzyme-product complex. On the basis of a 13-G line width in the EPR
signal of the flavin semiquinone (Fig. 6) observed in the two-electron
reduced cytochrome c reductase fragment is of the red
anionic type, as has been observed for native nitrate reductase
(21).
Given the difference in potentials between the two centers in the
cytochrome c reductase fragment of spinach nitrate
reductase, reduction of the cytochrome b by transfer of one
electron from FADH2 is highly favorable. Also, given the
expected proximity of the two centers (14), the intrinsic rate of
electron transfer is expected to be very fast compared to turnover and
not in any way rate-limiting. However, in the reaction of the
cytochrome c reductase fragment with NADH, electron transfer
is found to be relatively slow (12 s1), and the behavior
of the system is consistent with electron transfer being limited by the
breakdown of the charge-transfer complex between reduced flavin and
NAD+. Since dissociation of the product from the
FADH2·NAD+ complex is an obligatory step in
the course of turnover, this process likely represents the
rate-limiting step in overall catalysis of nitrate reductase, although
studies of the holo-nitrate reductase are required to definitively
establish this. Indeed, the 12 s
1 seen here for the
intermediate phase of the reaction (at 10 °C) compares fairly well
with the kcat of ~60 s
1 observed
with native spinach nitrate reductase under comparable conditions but
at 25 °C (33).
Previous studies with the cytochrome b reductase fragment of nitrate reductase have implicated the invariant cysteine 242 residue as playing an important but not essential role in the reductive half-reaction of the catalytic cycle (7). A comparison of the kinetic properties of the wild-type fragment with a C242S mutant indicated that the rate of reduction of the flavin decreased 7-fold on mutating the cysteine to a serine, and this was interpreted to mean that the cysteine played a role principally in facilitating electron transfer from NADH to the flavin (8). In light of the current results with the cytochrome c reductase fragment indicating that NAD+ dissociation is rate-limiting, it is possible that an equally important catalytic role of cysteine 242 is to facilitate NAD+ release from the charge-transfer complex formed at the completion of the fast phase of the reaction. It remains for future experiments to establish whether this is the reason that cysteine 242 is invariant among plant nitrate reductases.
We thank Dr. T. Suzuki and Prof. Toshi Yubisui, Department of Biology, Kochi University, Kochi, Japan for carrying out protein sequencing on the recombinant CcR and Craig F. Hemann for help with the circular dichroism studies.