Does the Reduction of c Heme Trigger the Conformational
Change of Crystalline Nitrite Reductase?*
Didier
Nurizzo
,
Francesca
Cutruzzolà§,
Marzia
Arese§,
Dominique
Bourgeois¶,
Maurizio
Brunori§,
Christian
Cambillau
, and
Mariella
Tegoni
From the
Architecture et Fonction des
Macromolécules Biologiques, UPR9039-CNRS, IBSM, 31, Ch. Joseph
Aiguier, Marseille Cedex 20, France, the § Dipartimento di
Scienze Biochimiche e Centro di Biologia Molecolare del CNR,
Università di Roma "La Sapienza," P. le Aldo Moro 5, 00185 Roma, Italy, and the ¶ ESRF, BP 220, 38043 Grenoble, France
 |
ABSTRACT |
The structures of nitrite reductase from
Paracoccus denitrificans GB17 (NiR-Pd) and
Pseudomonas aeruginosa (NiR-Pa) have been described for the
oxidized and reduced state (Fülöp, V., Moir, J. W. B.,
Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M. C., Mathieu, M.,
Cutruzzolà, F., Bourgeois, D., Fülöp, V., Hajdu, J.,
Brunori, M., Tegoni, M., and Cambillau, C. (1997) Structure
5, 1157-1171; Nurizzo, D., Cutruzzolà, F., Arese, M.,
Bourgeois, D., Brunori, M., Cambillau, C., and Tegoni, M. (1998)
Biochemistry 37, 13987-13996). Major conformational
rearrangements are observed in the extreme states although they are
more substantial in NiR-Pd. The four structures differ significantly in
the c heme domains. Upon reduction, a His17/Met106 heme-ligand switch is observed in
NiR-Pd together with concerted movements of the Tyr in the distal site
of the d1 heme (Tyr10 in NiR-Pa,
Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62
in NiR-Pa, 99-116 in NiR-Pd). Whether the reduction of the c heme,
which undergoes the major rearrangements, is the trigger of these
movements is the question addressed by our study. This conformational
reorganization is not observed in the partially reduced species, in
which the c heme is partially or largely (15-90%) reduced but the
d1 heme is still oxidized. These results suggest that the
d1 heme reduction is likely to be responsible of the
movements. We speculate about the mechanistic explanation as to why the
opening of the d1 heme distal pocket only occurs upon
electron transfer to the d1 heme itself, to allow binding
of the physiological substrate NO2
exclusively to the reduced metal center.
 |
INTRODUCTION |
The biological reactions involved in denitrification are not yet
completely understood at the molecular level. Bacteria, like Pseudomonas, Paracoccus, or Desulfovibrio, can
grow on nitrate as sole nitrogen source and can use nitrate as terminal
electron acceptor under anaerobic growth conditions (5). Four
elementary steps take place for the reduction of nitrate to
N2, catalyzed by nitrate, nitrite, nitric oxide, and
nitrous oxide reductase, respectively (6). Special attention has been
devoted to nitrite reductases
(NiR)1 because these enzymes
catalyze in vitro the four-electron reduction of dioxygen
with formation of H2O, as typical of membrane-bound terminal oxidases, and in vivo catalyze the electron
transfer from cytochrome c551 to nitrite with
production of NO. NiR-Pa is purified from the periplasmic space as an
homodimer of 120 kDa, carrying one c heme and one d1 heme
per subunit. The c heme is reduced first and the d1 heme is
the site of reduction of oxygen (7, 8) and nitrite (9, 10).
We have solved the structure of oxidized NiR-Pa (3). The d1
heme domain is an eight-bladed
-propeller in which a
-sheet of
four antiparallel
-strands forms each blade, and the c heme domain
has a typical class I cytochrome c fold, with a
His51-Met88 iron coordination. A peculiar
feature in NiR-Pa is the "domain swapping" (11, 12) of the
N-terminal region (6-29), which brings Tyr10 of the one
monomer close to the d1 heme site of the other.
Tyr10 is hydrogen-bonded by the OH side chain to a
hydroxide ion that is the sixth ligand of the d1 heme iron
and hinders the access of the catalytic site to the substrate. In NiR
from Paracoccus denitrificans GB17 (NiR-Pd) on the other
hand, the c heme has an unusual His17-His69
coordination in the oxidized state; moreover, the "domain swapping" is absent and Tyr25 of the c domain of the same monomer
directly coordinates the d1 heme iron (1).
Very unusual in the field of redox proteins, the NiR conformation
largely depends on the redox state of the protein, and major changes in
the structural organization were found to occur upon reduction. The
structure of reduced NiR-Pd recently published (2) shows large
rearrangements at the level of the c domain: the N-terminal arm from 26 (or 36) back is disordered after reduction, and in particular
Tyr25, a ligand of the d1 heme Fe(III) is
pushed out of the site upon reduction. Moreover, His17, one
of the two c heme ligands, is replaced by Met106 brought in
by the significant movement (15-17 Å) of the loop (99-116). In the
structure of the reduced NiR-Pa (NiR-red) (4), the rearrangements are
of smaller amplitude and consist of coordinated movements (Fig.
1): (i) a 6 Å rocking movement of the
loop (56-62) toward the c heme, likely stabilized by the formation of
new hydrogen bonds between Thr59 and Gln11 and
between Ala58 and Gly60, in the middle of this
loop, which tightens the loop in the reduced form; (ii) the rotation of
Tyr10 (4.2 Å) and the disappearance of the hydroxide ion
coordinating the d1 heme iron, which make the
d1 heme site accessible to the substrate. These movements
are independent of the presence of NO at the active site (4). Because
an identical movement of Tyr10 was also present when the
d1 heme was incompletely reduced, we proposed (4) that the
loop (56-62) displacement occurs upon reduction of the c heme and
triggers a cascade of movements.

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Fig. 1.
Stereo view of the conformational changes
associated with the change of redox state of NiR-Pa. The close
environment of the c and d1 heme in the oxidized
(thin line) and the reduced form (thick line) are
superimposed.
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In the present paper, we directly address the question of whether these
conformational changes are visible in an enzyme having the
d1 heme oxidized while the c heme is already reduced. With this aim, we have investigated the x-ray structure of NiR-Pa at different discrete levels of c heme reduction, all having the d1 heme oxidized. This experiment has taken advantage of
the slow intramolecular electron transfer from c heme to d1
heme in NiR-Pa, which has been already characterized in solution and
confirmed here in the crystalline state. The partially reduced forms of NiR-Pa were obtained by soaking the oxidized crystals in the presence of ascorbate as reducing agent and by cryoquenching the reaction at
different times. The redox state of the enzyme was assessed by
microspectrophotometry and the potential structural rearrangements being analyzed by the x-ray method.
 |
EXPERIMENTAL PROCEDURES |
Crystallization and Soaking Procedure--
The protein used for
the crystallization was purified following the method by Parr
(13). It had the expected absorbance ratio in the oxidized
state (A640 nm/A520 nm
1.0, A411 nm/A280 nm
1.2) and showed a single SDS-polyacrylamide gel electrophoresis
band. The crystallization conditions were described previously (3).
Orthorhombic crystals (P21212) were obtained in
2 M Na/K2-phosphate, 50 mM
Tris-HCl, pH 8.4, and had cell parameters of (163.1 × 90.1 × 111.9) Å (3). Oxidized crystals were soaked in a solution
containing 25 or 50 mM Na-ascorbate in 2 M
Na/K2-phosphate and 50 mM Tris-HCl, pH 7.2. Crystals NiR-15 and NiR-50 were soaked in 25 mM
Na-ascorbate for 6 and 30 min, respectively; whereas crystal NiR-90 was
soaked in 50 mM Na-ascorbate for 7 min. After soaking, the
crystals were transferred for a few seconds to solutions containing
15% glycerol. All the solutions were previously saturated with argon.
Microspectrophotometry--
In the microspectrophotometric
experiments, care was taken to record optical densities in the range of
linearity (OD
2.5). The ORIEL microspectrophotometer mounted on
the ID09 beamline at the ESRF consisted of a xenon lamp source and a
CCD detector. The spectra were recorded with unpolarized light. The
dark current corresponding to the electric noise was measured, and the
base-line correction was performed with a buffer similar to that in
which the crystals were soaked. The crystals were taken from the
soaking solution, mounted on a cryoloop, and frozen in a cold stream of nitrogen gas. The cryoloop put on the goniometer head was maintained at
100 K by a nitrogen cryostream (Oxford Cryosystems). Spectral processing (smoothing, displaying, and page setting) was performed with
the IDL5.1 (Research System, Inc.) and Xmgr3.01 (ACE/gr Development Team) software. The spectra of the frozen crystals were normalized at
541 nm, the isosbestic point in the transition between the oxidized and
the reduced form. On the normalized spectra, we calculated the
percentage of reduction of the c heme as the ratio of
OD to the
maximum
OD observed between the totally oxidized and the totally
reduced form at 545 and 551 nm in the
band of the c heme, where the
spectral contribution of the d1 heme is negligible compared
with that of the c heme. Three crystals (NiR-15, NiR-50, and NiR-90) at
15, 50, and 90% reduction of the c heme, respectively, were selected
for x-ray data collection. A control spectrum was recorded after the
exposure of the intermediate crystal at 50% c heme reduction. This
spectrum was not significantly different from that taken before
exposure to x-rays.
Data Collection, Processing, and Structure Refinement--
X-ray
data of NiR-15, NiR-50, and NiR-90 were collected at the ID14 beamline
at the ESRF (Grenoble, France) on a MarCCD detector, using a 0.933-Å
wavelength. A crystal to detector distance of 165 mm was chosen to
avoid spot overlap as much as possible. Data sets were indexed and
integrated with DENZO (14) and PrOW (15). They were merged with
the CCP4 suite (16) at a maximum resolution of 2.70 Å.
The NiR-ox model at 2.15-Å resolution (3) without any ions or water
molecules was used as the starting model. To accurately compare with
the NiR-red model, which had been refined between 12.0 and 2.90 Å (4),
the refinement of NiR-15, NiR-50, and NiR-90 was performed using the
same resolution range between 12.0 and 2.90 Å. Moreover, the same
reflection set was used for the calculation of the
Rfree, and an identical refinement procedure was
performed on all models and data sets (NiR-15, NiR-50, NiR-90, and
NiR-red). After several cycles of the rigid-body refinement procedure
in X-PLOR3.8 (17, 18), the two domains of each monomer were
satisfactorily fitted. At this stage, we calculated the first sigmaA
weighted maps (2mFo-DFc, difference Fourier with reduced model bias)
and (mFo-DFc) (19) (Figs. 3, A-C and 4A). The
refinement, including some conjugate gradient and B-factors refinement
cycles in X-PLOR3.8, was alternated with visual inspection on the
display, permitting the final model to be obtained. The refinement and stereochemical data are given in Table I,
in which the statistics show the good quality of the models despite the
absence of water molecules. The coordinates have been deposited at the
Protein Data Bank with codes 1N15, 1N50, and 1N90, respectively for the
NiR-15, NiR-50, and NiR-90 models and data sets. The r.m.s. deviations
were calculated with the LSQMAN program (20). Visual inspection and
manual rebuilding of the model were carried out with the graphic
program TURBO-FRODO (21).
 |
RESULTS |
Microspectrophotometric Characterization of the Reduction of NiR-Pa
Crystals by Ascorbate--
The spectral characterization of NiR-ox and
NiR-red in the crystal has been described previously (4). After the
addition of ascorbate to the mother liquor bathing the crystal of
NiR-ox, spectra between 700 and 400 nm were recorded. Upon reduction, the Soret band maximum shifted from 411 to 417 nm (not shown) and the
and
bands of the c heme become resolved; the maximum of the
d1 heme
band shifted from 640 to 650 nm. The crystals spectra, well resolved at low temperature, show the
band of the c
heme split in two peaks at 545 and 551 nm and the
band centered at
519 nm and flanked by two shoulders at 510 and 525 nm (Fig.
2).

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Fig. 2.
Optical microspectrophotometer spectra of
cryocooled crystals (100 K) between 480 and 700 nm. NiR-ox and
NiR-red (thick line), forms at approximately 15, 50, and
90% reduced c heme (thin line) and control of the 50%
after exposure (dotted line), respectively. The spectra were
recorded with unpolarized light. The crystal spectra have been
normalized at 541 nm, the isosbestic point in the ox-red
transition.
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When the crystals of oxidized NiR-Pa are exposed to Na-ascorbate 50 mM, the reduction of the c heme is fast and almost
complete in a few minutes, whereas the
band of the
d1 heme starts shifting toward 650 nm only after several
minutes (
10' at room temperature).
On the basis of the reduction time course observed in the crystal, it
seemed feasible to obtain crystals in which the d1 heme is
oxidized and the c heme is at intermediate levels of reduction. We have
then selected discrete times at which the reaction was stopped by
freeze-quenching the crystals. The redox state of NiR-Pa in the frozen
crystals was checked by microspectrophotometry before the x-rays
collection (Fig. 2); in particular, after having recorded and
normalized the spectra at 541 nm, we calculated the approximate proportion of the c heme in the reduced state. The spectra in Fig. 2
correspond to crystals in which the c heme is approximately at 15, 50, and 90% reduced. In all these spectra, within experimental error
limits, the
band of the d1 heme is superimposable to
that of the oxidized enzyme, showing that the d1 heme
remains oxidized throughout the reduction of the c heme.
The X-ray Structure of the Partially Reduced c Heme-Oxidized
d1 Heme Forms--
The crystals at various
percentage of the reduced c heme were suitable for x-ray data
collection and diffracted at the maximum resolution of 2.70 Å, which
is lower than that of the oxidized enzyme (2.15 Å) (3) but comparable
with that of the unliganded reduced enzyme (2.90 Å) (4). After the
rigid body refinement procedure using NiR-ox as a starting model and
the data sets of NiR-15, NiR-50, and NiR-90, and NiR-red crystals, we
calculated the first sigmaA maps (2mFo-DFc and mFo-DFc) (Figs.
3, a-c and 4a). We focused our attention
on the regions near the prosthetic groups where the wider structural
rearrangements had been observed in going from NiR-ox to NiR-red model
(4) (Fig. 1). At partial reduction of the c heme, the (2mFo-DFc) maps
are always clearly defined and fit well the oxidized conformation (Fig.
3, a-c). The conformation of the residues involved in the
movements (loop 56-62 and Tyr10) is superimposable to that
of NiR-ox. A peak in the Fourier difference map is observed in between
the two propionates of the d1 heme (Figs. 3,
a-c and 4a), which was successfully modeled with
a water molecule in the NiR-ox model (W564 and W65, in subunit A and B, respectively). Moreover, one additional Fourier difference peak is
observed between the iron atom of the d1 heme and the OH
side chain of Tyr10, with a shape comparable with that
observed in the NiR-ox structure at the same resolution and identified
there as an OH
ion (Fig. 3, A-C). No other
peaks are visible in the Fourier difference map, which accounts for the
good quality of the (2mFo-DFc) map. The presence of the
OH
strengthens the identity between the partially reduced
forms (NiR-15, NiR-50, and NiR-90) and the NiR-ox model.


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Fig. 3.
Stereoview of the electron density map
(2mFo-DFc, black; mFo-DFc, green) of
the partially reduced c heme forms (a, 15%;
b, 50%, and c, 90%). The model
of NiR-ox (balls and sticks representation in
atom-type colors) is superimposed to the electron density without any
changes.
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Fig. 4.
Stereoview of the electron density map
(2mFo-DFc, black; mFo-DFc, green) of
the reduced unliganded NiR-Pa. a, initial map with the model
of NiR-ox (balls and sticks representation in
atom-type colors) into the electron density without any changes.
b, same as in panel a after refinement.
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To truly estimate whether the absence of differences with the NiR-ox
structure is significant at the same stage of refinement, i.e. after the rigid body refinement procedure, we have
calculated sigmaA maps (2mFo-DFc and mFo-DFc) with NiR-ox as a starting
model and the data set of NiR-red (Fig. 4a). In this case,
the oxidized conformation does not fit the (2mFo-DFc) density map. The
clear (mFo-DFc) and (2mFo-DFc) maps appear showing the new tracing of the loop 56-62 that corresponds to the reduced conformation. Moreover, a peak in the mFo-DFc map appeared on the side of Tyr10,
likely indicating a displaced conformation of the side chain (Fig.
4a). A Fourier difference peak is also observed near the d1 heme but it is located between the proximal ligand
His182 and the iron atom of the d1 heme, far
from the position of the OH
in NiR-ox. This peak is
likely because of a misorientation of the His182 side chain
before refinement of the model. Indeed, after refinement by gradient
minimization and B-factor refinement, this difference peak has
disappeared, and the whole model has slightly moved around this
position. Both the loop 56-62 and the region of Tyr10 have
been rebuilt. After refinement, the new tracing corresponding to the
reduced conformation fit well into the density (Fig. 4b). In
the final model, two new hydrogen bonds are present between Thr59 and Gln11, and Ala58 and
Gly60, and a significant displacement of the loop 56-62
and Tyr10 with respect to the NiR-ox model can be observed.
The structures of partially reduced enzymes were superimposed on that
of the oxidized and of the reduced NiR-Pa, taking into account for the
r.m.s. calculation the backbone atoms of the d1 heme
domains. Based on this superimposition, the r.m.s. deviations were
calculated, and the results are presented in Tables
II and III.
The deviations to NiR-ox are in the range 0.32-0.56 Å; conversely those to NiR-red are 0.49-1.07 Å, values that are much closer to the
0.69-1.24 Å found for the difference between NiR-red and NiR-ox. By
taking into account only the residues involved in the conformational
change, in the active site and the ligands of the c and d1
heme groups, the r.m.s. deviations of the partially reduced heme c
forms versus NiR-ox are in the range 0.25-0.42, which is significantly different from the 1.4-1.7 found versus
NiR-red and for NiR-red versus NiR-ox (Table III). This
comparison strengthens the identity of the partially reduced c heme
forms to NiR-ox model.
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Table II
The r.m.s. deviation for the c heme domain (residues 30-115) and the
d1 heme domain (residues 150-536) calculated on the backbone
atoms of partially reduced NiRs (15, 60, and 90% c heme reduced) and
the fully reduced c heme, and NiR-ox or NiR-red, respectively, after
superimposition of the d1 heme domain of monomer A.
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Table III
The r.m.s. deviation of the atoms (258 non-hydrogen atoms) of the
residues involved in the conformational change, in the active site and
ligand of the c and d1 heme (residues 9-13, 54-64,
His327 and His369, His51, Met88,
His182, c heme and d1 heme) of partially reduced NiRs
(15, 50, and 90% c heme reduced) and the fully reduced c heme, and
NiR-ox or NiR-red, respectively, after superimposition of the
d1 heme domain of monomer A
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 |
DISCUSSION |
The present results on the reduction of crystalline NiR-Pa by
ascorbate are qualitatively in agreement with the characterization of
the reduction reaction previously carried out in solution in the
presence of reduced azurin, in which the c heme is reduced more rapidly
with a rate constant value k1 = 28 s
1 at room temperature (22), whereas the intramolecular
electron transfer is 35-fold slower in the absence of an external
ligand acting as an electron acceptor (23, 22). A faster intramolecular electron transfer was detected in the reaction with O2,
where the c heme is oxidized at a rate constant
k2
100 s
1 (8). The
quantification of the reduction kinetics in the crystal would demand a
complete characterization of the reaction time course, which has not
been carried out because it was not the primary motivation of the
present study; our goal was to produce and freeze NiR-Pa crystals at
different levels of reduction of the c heme to estimate the sequence of
structural events involving the d1 heme active site.
NiR-Pd crystals have previously been characterized by time-resolved
microspectrophotometry during the reduction by dithionite and the
reoxidation by nitrite (2). An accurate analysis of the spectra
published in Williams et al (2) indicates that, in contrast
to the reaction of ascorbate with NiR-Pa, no significant lag was
detected between the reduction of the c and the d1 heme. Indeed, a fast intramolecular process has also been observed in a pulse
radiolysis study of NiR-Pd in solution; the rate constant of 1400 s
1 at pH 7.0 and room temperature was assigned to the
electron transfer from the c heme to the d1 heme (25).
The x-ray data presented above on the intermediate oxidation states of
NiR-Pa indicate that the structural modification at the level of the c
and the d1 heme observed in going to the fully reduced
NiR-red are absent in the partially reduced NiR-15, NiR-50, and NiR-90
models. In light of these results, and in contrast with our previous
hypothesis suggesting that the loop 56-62 might move upon reduction of
the c heme and trigger the concerted movements at the level of the
d1 active site (4), our present interpretation is that the
reduction of the c heme by itself does not trigger the movements of the
loop 56-62 and of Tyr10; these conformational changes
appear therefore to be a consequence of additional events in the
reduction process that are likely to be the electron transfer to the
d1 heme iron.
From the results in the literature and our studies, the catalytic cycle
of NiR-Pa can be summarized by the following Scheme 1.

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Scheme 1.
Catalytic cycle of NiR-Pa. Step 1, [1] [2]: bimolecular 1.5 × 106
M 1 s 1 in the presence of Azurin
as electron donor (23); monomolecular 28 s 1 with Azurin
in excess (22). Step 2, [2] [3]: 0.8-1.0 s 1
intramolecular electron transfer from c heme to d1 heme
(10, 22, 23). Step 3, [3] [4]: supposedly identical to step 1. Step 4, [4] [5]: producing NO and H2O; 108 M 1 s 1 (10).
Step 5, [5] [2]: non-determined. Step 6, [5] [6]: as slow
as step 2. Step 7, [6] [7]: faster than step 1 (10).
[1], characterized by microspectrophotometry and crystal
structure; [2], characterized microspectrophotometry and
crystal structure; [3], transient in the catalytic cycle;
[4], characterized by microspectrophotometry and crystal
structure; [5], transient; [6], transient in
the cycle at pH > 7; [7], NO complex characterized
by microspectrophotometry and crystal structure (complex with NO
blocked at basic pH). Crystal forms in the reduced conformation
(d1 heme site open) are cross-shaded, whereas
forms in the oxidized conformation are
straight-shaded.
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For step 1, [1]
[2], the c heme of the c-ox/d1-ox
form is rapidly reduced by the electron donor cytochrome
c551 leading to the c-red/d1-ox
form. The preference of this electron donor for the c heme can
originate for two reasons: either the potential of the c-heme is more
positive than that of the d1 heme (26) or the c heme is
more accessible than the d1 heme (27). The latter is
confirmed by the three-dimensional structure of NiR-Pa.
For step 2, [2]
[3]
[4], the c-red/d1-ox form
goes through a step involving electron transfer and coupled
conformational changes, although the transient c-ox/d1-red
form was not isolated, being immediately followed by the
c-red/d1-red form in the presence of excess reductant. The
step [2]
[3] is slow and might be limited by a slow electron
transfer or a slow conformational change involving a reorganization of
the coordination site. The d1 heme reduction might be the
trigger of the conformational change. As it is generally agreed that
the affinity of oxygenated ligands is much greater for Fe(III) than for
Fe(II) (28), addition of one electron at the Fe center of
d1 heme (its reduction) might weaken the
Fe-OH
bond and provoke the release of the hydroxide ion.
This would then induce a series of conformational changes involving the
displacement of Tyr10, likely attracted by the
His369 to which it is hydrogen-bonded (2.7 Å in NiR-ox),
coupled to the motion of the loop 56-62 of the c domain. In line with
this hypothesis, in mutants of sperm whale myoglobin in which the
distal His64 had been changed to Tyr, the coordination bond
between the OH of Tyr and the Fe was weakened or abolished upon
reduction (29). Alternatively a redox state-dependent
change of the pK of His369 might be responsible for a
shortening of the hydrogen bond between this residue and
Tyr10 (2.7 Å in NiR-ox) and a weakening of the
Tyr10-OH
ion hydrogen bond. Indeed, the
subunit B of the unliganded NiR-red (4), where the d1 heme
is partly oxidized, presents a conformation close to that proposed
above because the Tyr10 is in the rotated position, with
the OH at 3.45 Å from the N
2 of His369, and the
Fe(III), as we proposed (4), is coordinated to an hydroxide ion. In
accord with this second hypothesis, in the H64Y mutant of sperm whale
myoglobin, a pK of 5.6 for Tyr64-OH has been suggested to
depend on the close proximity of basic residues (30). However, for
NiR-Pa an altered kinetic behavior would have been expected for the
mutants Y10F and Y10N, in which the hydrogen bond to the hydroxide
should not be formed (22).
For step 3, [4]
[5]
[2], the c-red/d1-red form
binds nitrite and reduces it to nitric oxide. In the process, the
d1 heme becomes oxidized. At low pH, the affinity of the
d1-oxidized heme for NO is low (
1 mM) (9),
and NO is therefore released ([5]
[2]), leading to the
c-red/d1-ox free enzyme, ready to initiate the next cycle.
When the pH is basic, NO remains bound to the c-red/d1-ox enzyme long enough to lead in the presence of reductants to the stable
c-red/d1-red-NO form which is inactive and essentially "trapped" (10). The protonation state of the active site
histidine(s) (His369 and His327) might be
responsible of the high affinity of NO for the enzyme at basic pH.
 |
Conclusion |
An intriguing question is why a conformational change occurs
during catalysis. It is first of all clear that the structure of the
reduced enzymes from NiR-Pa and NiR-Pd are very similar at the level of
the c heme, including the conformation of the loop 55-60. This
conformation is also that found in cytochromes c and should
therefore be rather stable. The role of the conformational change of
this loop in catalysis remains unclear but might suggest that
prevention of a free access of ligands to the d1 active
site is necessary. Indeed, the Tyr movement might help the opening of
the d1 active site at the proper step in catalysis when the reduced d1 heme should bind the physiological substrate
NO2
and avoid the formation of catalytically
incompetent complexes, like Fe(III)-NO2
. The
presence of this conformational change at the d1 heme
active site might be explained by the need of populating a "resting
state" in these enzymes to make the d1 heme inaccessible.
In both these enzymes, the inaccessibility of the heme Fe in the
d1 site might be assured either by direct coordination of
Tyr25 in NiR-Pd or by the intervention of
OH
/Tyr10 in NiR-Pa. Some analogies in the
structure of NiR active site and eukaryote cytochrome c
oxidase might be envisaged, as in both cases a Tyr stabilizes an
oxygenated ligand (OH
and O2) coordinated at
the Fe of the heme (d1 and a3) (24). In
cytochrome oxidase, nevertheless, it seems that the protonation state
of the Tyr triggers the shift to a catalytically active state of the
enzyme, whereas in NiR the role of Tyr seems to protect the active site
when the enzyme does not turn over and is, in this respect, somewhat
analogous to the CuB of cytochrome c oxidase.
 |
ACKNOWLEDGEMENTS |
The ESRF is greatly acknowledged for beam
time allocation (ID14). We thank Alain Desbois for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by a EU BIOTECH Structural Biology
project (BIO4 CT96-0281) and by MURST of Italy (Programma Nazionale Biologia Strutturale 1997).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1N15, 1N50,
and 1N90) have been deposited in the Protein Data Bank, Brookhaven
National Laboratory, Upton, NY.
To whom correspondence should be addressed. E-mail:
tegoni{at}afmb.cnrs-mrs.fr or cambillau{at}afmb.cnrs-mrs.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
NiR, nitrite
reductase;
NiR-Pa, nitrite reductase from Pseudomonas
aeruginosa;
NiR-Pd, nitrite reductase from Paracoccus
denitrificans;
NiR-red, reduced NiR;
NiR-ox, oxidized NiR;
r.m.s., root mean square.
 |
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