(Received for publication, May 18, 1995; and in revised form, August 23, 1995)
From the
High resolution x-ray crystallographic structures of nitrite
reductase from Achromobacter cycloclastes, undertaken in order
to understand the pH optimum of the reaction with nitrite, show that at
pH 5.0, 5.4, 6.0, 6.2, and 6.8, no significant changes occur, other
than in the occupancy of the type II copper at the active site. An
extensive network of hydrogen bonds, both within and between subunits
of the trimer, maintains the rigidity of the protein structure. A water
occupies a site 1.5 Å from the site of the type II copper in
the structure of the type II copper-depleted structure (at pH 5.4),
again with no other significant changes in structure. In nitrite-soaked
crystals, nitrite binds via its oxygens to the type II copper and
replaces the water normally bound to the type II copper. The
active-site cavity of the protein is distinctly hydrophobic on one side
and hydrophilic on the other, providing a possible path for diffusion
of the product NO. Asp-98 exhibits thermal parameter values higher than
its surroundings, suggesting a role in shuttling the two protons
necessary for the overall reaction. The strong structural homology with
cupredoxins is described.
Nitrite reductase (EC 1.7.99.3) from Achromobacter cycloclastes is a copper-containing protein that reduces nitrite to NO in .
This reaction is the second step in a dissimilatory pathway (1) in which N is the ultimate produc
t. This step
requires donation of electrons to NIR (
)by another copper
protein, pseudoazurin(2, 3, 4) .
The
structure determination of A. cycloclastes NIR at 2.3-Å
resolution (5) showed that the molecule is a trimer, with a
total of six copper atoms/trimer, and that each monomer contains two
domains that fold into Greek key -barrels similar to those of
cupredoxins such as plastocyanin and pseudoazurin. Each monomer has a
type I copper (bound by His-95, Cys-136, His-145, and Met-150) and a
type II copper bound by His-100 and His-135 from one monomer and
His-306 from a second monomer. A water molecule is the fourth ligand to
this copper. Preliminary crystallographic studies (5) indicated
that the type II site is the site at which nitrite interacts. The
specific activity of the protein correlates with the amount of copper
at the type II site (6) .
NIRs with 80% sequence
similarity have been isolated from four organisms: A.
cycloclastes(7) , Alcaligenes faecalis S-6(8) , Pseudomonas sp. G-179(9) , and Pseudomonas aureofaciens (10) (Fig. 1). The x-ray
structure of A. faecalis S-6 NIR has recently been extended to
2.0 Å (
)and is much the same as that of A.
cycloclastes NIR(5) . A fifth NIR, from Alcaligenes
xylosoxidans, also has the same fold, as shown by low angle x-ray
scattering studies(13) , and also exhibits dependence of
specific activity on the amount of type II copper present (14) . Recent electron nucleus double resonance studies on A. xylosoxidans NIR have demonstrated
NO
binding to the type II
copper(15) .
Figure 1: Sequences of four copper-nitrite reductases. Conserved residues are boxed. Ach, A. cycloclastes (7); Af, A. faecalis (8); Psp, Pseudomonas sp. G-179 (9) ; Pau, P. aureofaciens(10) . The first three appear more similar to each other than to the fourth.
Denitrification may not be found only in bacteria. A denitrification pathway has been found in the mitochondria of the fungus Fusarium oxysporum and shown to contain a copper-nitrite reductase that makes NO and that utilizes an azurin or cytochrome c as an electron donor(16) . Recent reviews describe other components of the bacterial pathway(17, 18) .
Site-directed mutagenesis of A. faecalis NIR has shown that the type I site mutant, M150E,
can no longer be reduced by pseudoazurin, while the type II site
mutant, H135K, can no longer reduce NO.
These results suggest that the path of electron transfer is from
reduced pseudoazurin to the type I copper to the type II copper and
then to nitrite(12) . A plausible model for the interaction of
pseudoazurin and NIR has been proposed from the observation that
pseudoazurin has a ring of positively charged lysines surrounding the
hydrophobic surface through which the histidine ligand to the type I
copper protrudes and that NIR has a somewhat less extensive ring of
negatively charged residues surrounding a similar face near its type I
copper center. When lysine residues close to the hydrophobic surface
are mutated individually to Ala, the K
for interaction with NIR increases. When lysine residues far
from this surface are similarly mutated, the K
remains unchanged(19) .
A pH optimum of 6.2 for
the reaction with nitrite has been observed for both A.
cycloclastes NIR (20) and A. faecalis NIR (2) . There are two residues near the type II copper ligands,
His-255 and Asp-98, that are between the active-site copper and the
rest of the solvent. We hypothesize that these might be the immediate
source of protons in formation of the product water. To provide data to
see if this is the case and to understand the pH profile, we have
collected diffraction data at five different pH values: 5.0, 5.4, 6.0,
6.2, and 6.8. The first three are from crystals with a cubic unit cell
reported previously(3) , the last two are from orthorhombic
crystals(21) . We have carried out refinements of the cubic and
orthorhombic structures as well as the type II copper-depleted
structure(6) . Finally, we have extended our analysis of
nitrite-soaked crystals and describe the NO complex with NIR. An extensive structural description is provided
for future site-directed mutagenesis studies and other anticipated
comparisons.
The type II copper-depleted NIR (T2D) was
prepared as described previously(6) . The EPR spectrum of the
T2D sample used for crystallization indicates there is <10% type II
copper present (data not shown). ()Crystallization of the
still green (consistent with the notion that the type I copper is the
chromophore) material was carried out as for the native protein. The pH
of the crystals was taken as the nominal pH of the buffers used for
crystallization, without added ammonium sulfate.
Figure 4: MOLSCRIPT drawings of a monomer of NIR, with residues defining secondary structure labeled. a, view approximately from the interior of the trimer; b, rotated 180° about the vertical axis.
Figure 6: MOLSCRIPT drawings of three layers (a-c) of the charge interactions near the 3-fold axis of the trimer, from top to bottom of the channel. Not shown is a phosphate near residue 251 in the KP structure.
Figure 7:
Difference maps illustrating variable
occupancy of copper in the type II site. The solvent channel extends to
the right from the orientation shown. Phases are from T2D; all maps are
at 2.0-Å resolution for comparison (except those involving NT1).
The locations of the water bound to copper (Cu-OW) and that
bound in the absence of copper (T2D-OW) are shown. Positive contours
are solid lines, and negative contours are dashed
lines. The differences seen in a suggest that HR has more
Cu
than PH5, and PH5 has a more ordered Asp-98. PH5 and KP
have almost the same amount of copper and no difference in Asp-98
motion. HR has a more complex difference at the OW sites, as does
NT1.
Figure 8:
Difference maps illustrating differences
from T2D data, showing that there is indeed little copper in the
Cu site in T2D and that there are differences at the OW
site as well. Positive contours are shown as solid lines, and
negative contours are shown as dashed lines. Orientation and
phases are as described for Fig. 7. d suggests that
Asp-98 is more ordered in the T2D structure than in the NT1
structure.
Figure 9:
F - F
maps for T2D (a), PH5 (KP and
HR) (b), and NT1 (c and d). In each, T2D-OW,
Cu
-OW, or NO
was omitted from the F
calculation. a-c are
oriented as described for Fig. 7and Fig. 8, while d is oriented 90° around the vertical axis for clarity. Here, it
can be seen that the plane of His-255 is oriented such that the proton
on N
will hydrogen bond to OW-1098 (seen only as a
sliver just under the HIS 100 label) and not to either T2D-OW
or Cu
-OW. It can be seen that in a, T2D-OW is
closer to Cu
than Cu
-OW is in b.
Standard positional refinement and temperature factor (B) refinement were carried out in separate steps. Solvent molecules were located by systematic peak searches of difference maps and examined on the graphics before accepting for refinement. Usually, these peak searches first revealed well ordered solvent, then alternate side chain conformations, and finally misplaced side chains. Solvent was eventually labeled by the code nyyy, where n = 1, 2, 3 . . . and yyy is the residue number of the closest amino acid, facilitating subsequent comparisons.
The initial multiple isomorphous replacement model (5) was refined using 10 to 2.3-Å data. Starting B-values represented estimates of the quality of the density fit by that residue. The R-value decreased from 0.46 to 0.296 with one round of positional refinement and one round of rebuilding from difference maps, followed by another cycle of positional refinement. A run of simulated annealing led to an R-value of 0.254. At this point, a data set was collected from an orthorhombic crystal.
Several rounds of refitting and positional refinement including three rounds of B refinement resulted in an R-value of 0.187 for data from 10 to 2.7 Å. At this point, it was noted that the geometry at the copper sites was not consistent, and distance restraints were tried, to little avail. Data to 1.6 Å from the type II copper-depleted cubic form became available, and it was decided to proceed with the higher resolution refinement and to complete the other models using those results.
Figure 2:
R-factor versus resolution
(expressed as sin/
) for the seven
structures. The dotted lines indicate expected coordinate
error (in angstroms) given only random errors remaining in the
coordinates. From this plot, one would conclude that the overall
coordinate error is on the order of 0.18 Å for the T2D structure
and 0.2 Å for the others.
Figure 3: B-value versus residue number for four structures. Main chain averages are above the horizontal lines, and side chain averages are below. B-values for 68 are like those for 62; those for KP and HR are like those for PH5 and thus are not shown. It is likely that the unusually low values for 62 are the result of still inadequate scaling. The undulating patterning is clearly the consequence of the secondary structure in the protein.
The
protein exhibits normal conformations for most residues; >90% fall
in the most favored regions of the -
plot, and the
remaining are in allowed regions. Fifteen to twenty deviations from
ideality for N-C
-C (
) angles are tabulated by
X-PLOR and PROCHECK(33) . Several are associated with glycines
(Gly-222, Gly-261, and Gly-268). Deviations associated with large
hydrophobic residues (Ile-218, Phe-270, and Phe-312) indicate an
opening up of that angle
4-5 times the root mean square
value. Ile-218 is involved in a twisted pair of
-strands that
interacts with a topologically similar pair from domain 1 of the same
molecule. Phe-270 is in a loop remote from the active site. Phe-312 is
part of a small helical loop between the two strands of
-sheet at
the C terminus of the molecule. It forms an important part of the
packing interaction between monomers as well as part of the hydrophobic
wall of the active-site cavity.
angles of Tyr-303, Asn-305, and
Asn-307 are smaller by 5-8 times the root meant square deviation,
or
96° instead of the usual 111°. Tyr-303 is the last
residue in the
-strand, which then turns into the short helix
initiated at His-306.
angles for Tyr-303, Val-304, Asn-305,
His-306, Asn-307, and Leu-308 are 100°, 116°, 95°,
121°, 95°, and 120°, respectively, by far the largest
deviations seen in the structure. The
angle for His-306 is also
significantly different from normal (165°). Residues with larger
angles face into the active site, while those with smaller angles
in this group do not. The net effect of the alternation of angles is to
compress the surface facing into the active site and to slightly expand
the other side.
An interesting exception to the usually observed correlation of B-value with secondary structure (Fig. 3) occurs in the active-site cavity: Asp-98 invariably appears in negative density in difference maps, suggesting that the smoothly increasing B-value restraint put on this side chain does not adequately model the side chain. The B-values of the copper atoms are generally higher than those of their ligands (see Table 4), which is unusual for fully occupied metal sites. Although not refined, we estimate that the occupancy is no less than 0.8 to account for the B discrepancy.
As noted in Table 3, several residues have alternate
conformations. These residues are mostly charged surface residues, but
several valines also exhibit alternate conformations. Val-17 and Val-20
are in the extra N-terminal loop, which appears to be only loosely
attached to the main body of the -barrel. Val-146 is in loop
136-150, containing three of the ligands for the type I copper,
and has little to constrain it to a preferred orientation. It is
adjacent to Val-142, in a helix, but is not involved in packing between
domains to the same extent as Val-142. Val-237 is at the end of a
-strand and is exposed, but other valines are also exposed and not
distinctly disordered. No residues interior to the
-sheets are
disordered.
Figure 5:
Topology diagram of nitrite reductase. a, domain 1; b, domain 2. Residues involved in
intradomain hydrogen bonds are designated by black square
outlines; ligands are indicated by black circular
outlines. W, solvent; X, side chain; O and N, main chain atoms. Leftward diagonally striped
circles indicate conserved residues; rightward diagonally
striped circles indicate similar residue types. Pentagons denote crystal contacts for the cubic form, and upside-down
pentagons indicate those for the orthorhombic form. Topology is
drawn as if from the outside of the -barrels. Doglegs appear as breaks between sheets.
In the loop connecting the next two strands to the back in Fig. 4a are residues 66-70, which define the dogleg in domain 1. In domain 2 (right-hand domain in Fig. 4a), residues 220-232 form a ``double'' loop dogleg between the N- and C-terminal sheets. (If the two domains are superposed, the dogleg of domain 1 superposes with the first five residues of the dogleg in domain 2.) In domain 2, the double loop forms an edge into which fits the raised edge from domain 1 formed from residues 68-70 and 20-23. Together, the first parts of each dogleg form, along with residues 187-200, a crevice into which neatly fits a folded-back (and twisted) N-terminal loop (residues 51-58), somewhat like how the tongue on a belt buckle rests on the buckle. This folded-back loop corresponds to a folded-back loop in pseudoazurin, which contributes to an extensive hydrophobic surface, believed to interact with NIR(19) , through which a type I copper ligand protrudes. Interestingly, the topologically corresponding loop of domain 2 (residues 187-213; the ``tower'') in NIR is also probably involved in interacting with pseudoazurin.
One of three
nearly helical regions of the structure is in the tower, which connects
10 and
11 and provides interdomain interactions with the
topologically analogous loop connecting
1 and
2 in the first
domain described earlier. The only other helical regions are
, which provides three ligands to the type I copper in
domain 1, and its topological counterpart in domain 2,
, which provides some of the residues lining the
active site as well.
Residues 160-171, connecting the two domains, are rather loosely suspended in a loop across a gap that could be spanned equally well with residues 162-170 clipped out. However, these two short antiparallel strands may fill in a crevice and stabilize the protein.
The interior of both domains is largely
hydrophobic. The ``bottom'' half of domain 2 is filled with
optimally packed aromatic residues (Tyr-178, Phe-220, Phe-292, Tyr-301,
Tyr-303, Phe-320, Phe-295, and Phe-263). Included in this is the Tyr OH
X
loop (Tyr-301 OH to
Gln-297 nitrogen and oxygen), common to most Greek-key
-barrels.
Domain 1 has a phenylalanine (residue 132) in that position.
The top of domain 2 is the structural analog of the type I copper region in domain 1 and is filled with a remarkable series of buried side chain-side chain hydrogen bonds, starting with Trp-281, which extends to residues near the 3-fold axis (Fig. Z1). (These residues are marked by filled black square outlines in Fig. 5b.)
Figure Z1: Structure 1
The deeply buried waters, positions 1303 and 1180, are in the group
of 30 solvent molecules with B-values <25 and must be
incorporated while the protein folds as they are so buried. This
extensive internally hydrogen-bonded region leads to the top of the
barrel where the strands have splayed out somewhat (the expanded, not
compressed, side of the
-sheet described earlier). In domain 1,
the only interior hydrogen-bonded interaction is the conserved
His-95-Asn-47 interaction, which serves to orient the type I
copper ligand, His-95, analogous to such interactions found in
cupredoxins.
There are only nine hydrogen bonds between protein atoms of domain 1 and atoms of domain 2 (Table 7, part c). All except the ones involving Val-131 are between the tower and the ``twisted loop''/dogleg interaction described above. Of nine solvent-mediated interactions between domains 1 and 2 (Table 7, part d), two involve solvent with low B-values (Fig. 5, open diamonds).
Subunit
interactions are of two kinds: interactions close to the trimer axis
and those participating directly in formation of the active-site
cavity. The interactions around the trimer axis, containing only
residues from domain 2 (Fig. 6, a-c), consist of
three layers of alternating charge. Not shown are one hydrophobic
contact closing off the channel (Leu-282) and several solvent molecules
on the 3-fold axis as well as surrounding it. The KP structure contains
a sulfate (or phosphate) on the 3-fold axis, near Asp-251. Alternate
conformations noted in Table 3for Arg-250 (a rotation around the
C-C
bond) do not alter this
charge distribution. A remarkable feature of formation of the
active-site cavity is the extensive antiparallel hydrogen-bonded
interaction of the C-terminal tail with residues 117-125; other
features local to the cavity are described below in conjunction with
the type II copper site.
The solvent structure is fairly typical,
with 0.5 solvent molecule/residue. The distribution of solvent B-values is bimodal, with a peak at
20 Å
and another near 45 Å
. All of the solvent
molecules with B <25 Å
are found
internally, between domains, or between subunits in the active site
(marked in Table 7, parts d, f, and g). All of these have two or
more interactions with protein and must be considered an integral part
of the protein structure.
There are a few reorientations of side chains (e.g. residues 54 and 168) among the structures, but these are attributable to differences in modeling of some high thermal parameter side chains. The trimer is a well constructed object resistant to change with pH or the presence or absence of copper and/or the substrate at the active site. It has many features that serve to maintain its overall integrity, even in spite of unraveling of residues 8-10 at the N terminus and residues 336-340 at the C terminus of monomer A to form the new crystal contacts of the orthorhombic form.
As in
pseudoazurin, a number of features in the protein serve to provide the
rather rigid framework for these ligands. There is one NH-S
hydrogen bond to Cys-136, from the amide nitrogen of residue 96. Since
residue 138 is a proline, as in plastocyanins, the second NH-S
bond found in azurin is lacking here as well. However, in contrast to
plastocyanin, the proline has a markedly different position and even if
mutated to an alanine would not provide an amide hydrogen directed
toward the cysteine; this results from the additional four residues
between the two copper ligands, Cys and His, relative to plastocyanin.
The additional residues protrude from the N-terminal end of and form part of the active-s
ite cavity.
These additional four
residues between ligands also affect a heretofore conserved Asn
Ser/Thr interaction near the type I site. The side chain of Asn-96
forms a pair of hydrogen bonds with N-137 and O-137, maintaining an
extended peptide orientation adjacent to the cysteinyl copper ligand,
as is always found in the analogous region in cupredoxins. However, in
cupredoxins, a conserved serine or threonine just following the
cysteine provides two hydrogen bonds, one between O
of
the Asn and NH of the serine or threonine and the other between
N
and O
of the serine or threonine.
Here, instead, N
hydrogen bonds to the carbonyl oxygen
of the alanine.
His-95 is oriented by the side chain of Glu-47,
which in turn is also hydrogen-bonded to the amide nitrogen of Thr-92.
His-145 is more buried than its counterparts in pseudoazurin, largely
occluded by Trp-144, Met-141, and Leu-93 and making unlikely a
titratable His ligand as seen in pseudoazurin, amicyanin, and
plastocyanin. Nevertheless, like the pseudoazurins, this exposed edge
of His-145 is in the midst of a hydrophobic surface, with Asn-115 and
Gln-113 being the closest solvating groups. Surrounding the hydrophobic
surface is an arc of negative charge, similar to that described for A. faecalis NIR. Met-150 is sandwiched between
Met-62 and Phe-64, just as its counterpart in pseudoazurin is also
buried between two large hydrophobic residues.
We have previously
noted (36) that the copper in NIR is farthest from the plane
(0.4 Å) of the three strongest ligands for any of the type I
sites. It was suggested that the larger number of residues between
copper ligands in the C-terminal loop might contribute to this.
Superposition of the NIR type I site and the pseudoazurin type I site
shows that the extra residues protruding from the N-terminal end of the
short helical loop might in fact form a slightly larger box for the
copper to be in, but as is the usual case, there is not a simple
explanation for the phenomenon or for the shorter copper-Met
distance.
Interpretation of these difference maps is complicated
by the fact that the T2D data clearly show that when copper is not bound, a water is found at the site (Fig. 9a). The
10% copper estimated to be in our sample from EPR data is detectable as
density at the copper site when T2D-OW is included in the model. The
water is not precisely at the copper position, but displaced
from it 1.6 Å toward solvent (0.5 Å from the water ligand
site; see Fig. 7Fig. 8Fig. 9) such that reasonable
hydrogen bonds can be made from two of the histidines and to Asp-98
(distances: OW-His-100 N, 2.96 Å;
OW-His-135 N
, 3.04 Å; and OW-Asp-98
O
, 2.77 Å; angles: 100-OW-135, 97°;
100-OW-98, 80°; and 135-OW-98, 112°). His-306 is 3.2 Å
away and on the same side of the water as the other three residues, so
is not likely to be hydrogen-bonded. The possible fourth coordination
site is blocked by Ile-257 and the remarkably apolar environment in
that region. The histidines do not change position at all. When copper
is not present, one histidine must be protonated (probably His-306) and
at least one unprotonated in order to accept or donate hydrogens to the
water and to provide the same overall formal charge as the copper
(2+ before reduction).
The water bound to the copper is most
clearly seen in the KP-T2D map (Fig. 8b), where density
for the water is most well resolved. Fig. 9shows F - F
difference maps
for each of the data sets, with the water ligand not included in the F
calculation. In the refinement, the water
modeled at the active site generally shows at least as low a B value, at full occupancy, as does the copper. The difference
density for PH5, HR, and KP exhibits perceptible differences at the
water site. The KP water position is most spherical, while that for PH5
and HR exhibits nonspherical shape, which could be modeled by a
combination of the water site when copper is absent and the water site
when copper is present, consistent with partial occupancy of the
copper. The Cu-O distance of
1.9 Å (Table 8) is
comparable to the Cu-H
O distances in small compounds
(1.96-2.41 Å in CuSO
5H
O and
2.0 Å in CuH
O(37) ).
Nitrite replaces the water ligand and binds to copper via the two oxygens, one at 2.4 Å and the other at 2.1 Å (Fig. 9, c and d). Since the density is larger at the water ligand site, there must be partial occupancy of only water in some of the molecules in the crystal. The nitrite has been modeled in the structure at 0.5 occupancy.
The three histidine ligands are nearly equidistant to the
type II copper: N-Cu is 2.05 ± 0.1
Å over all of the structures. The angle between His-306, copper,
and His-135 is consistently larger (111° versus 101°)
than that between His-306, copper, and His-100, while
His-100-Cu-His-135 is
104° (Table 9). The
Cu
water is not perfectly tetrahedrally disposed, but quite
close to it.
Figure 10:
MOLSCRIPT drawing of the active-site
pocket with nitrite bound, illustrating the clown face appearance as
viewed from solvent into the active site. a, only domain 2 of
the left-hand molecule (B) is shown; domains 1 and 2 of the
right-hand molecule (A) are shown, with domain 2 darkened relative to domain 1 in order to see its contribution to the back
side of the active site. b, close-up of the active site with
nitrite bound, showing the relationship of His-255, Asp-98, OW-1098,
and nitrite to Cu. The hydrophobic surface on which NO is
postulated to diffuse out is on the left.
The residues lining the left-hand surface of the pocket are all hydrophobic (Leu-308, Phe-312, Val-304, Ala-302, Ala-317, His-255, and Ile-257), except for His-255 at the back, while those on the right side are more hydrophilic (Leu-106, Gly-109, Gln-113, His-100, Asp-98, Asn-96, His-135, and Glu-139). No waters are found on the left-hand side, whereas several ordered waters are found on the right as well as in and along the seam lacing together the lower interface (where the palms meet).
The two halves of the
region defining the clown face are related by a pseudo 2-fold axis
(also described in the A. faecalis NIR structure).
The lower right of the front of this surface is composed of conserved
residues 106-111 (LGGGAL) forming a small irregularity on the
edge of a
-sheet. Its 2-fold counterpart, residues 255-260
(HLIGGH), is also conserved and forms an irregularity on the edge of
its
-sheet. These short conserved stretches appose Leu-106 and
Ile-257 at the lower entrance to the pocket, one point on the pseudo
2-fold axis. A second defining point is the midpoint between His-306
and His-135. This 2-fold axis relates the two domains of different
molecules in the same way that a pseudo 2-fold axis relates the two
domains forming the active site of ascorbate oxidase(11) .
Interestingly, while residues 255-260 are part of the hydrophobic
wall of one active site, residues 245-250 on the same strand form
part of the back wall of the next active site.
The back of the pocket is formed by loops 284-288 and 246-250 of domain 2 of the right-hand molecule (monomer A), as viewed from the outside into the active site. If this domain were absent in this subunit, there would be access to the copper from the other side of the active site that related by the pseudo 2-fold axis to the open access channel. Domain 1 never contacts another symmetry mate around the 3-fold axis, whereas domain 2 does.
Connecting loops 247-250 and
284-288 not only form the back of the pocket, but also make an
unusual environment for the two His ligands, His-306 and His-100.
(His-135 N-H is pointed toward Cys-136 carbonyl oxygen
(with a N-O distance of 3.0 Å).) His-306
N
-H points midway between O-306 and O-248, the latter
being constrained by a conserved QAN sequence. Similarly, His-100
N
-H is pointed toward two oxygen atoms, O
of Glu-279 (in turn oriented by Lys-269) of a neighboring monomer
in the trimer and the oxygen of Gly-286 in its own monomer. Each of
these composite interactions is formed from crevices between domains.
The first loop, 247-250, also provides an essential residue in
the interactions around the 3-fold axis (Fig. 6), where Arg-250
alternates with Glu-310 to form one layer of alternating charge, and
Asp-251 and Arg-253 form another just above, lacing up the trimer. The
sequence SQANRDXR (residues 246-253) is conserved among
the four known NIRs.
Aside from variable copper occupancy and some small packing
interactions, there is little difference among these structures as the
pH is changed from 5.4 to 6.8, or the copper removed, or nitrite added.
A large number of hydrogen bonds provide a very robust framework for
this protein. Surface area calculations on the closely related A.
faecalis NIR show that 28% of the monomer surface
(
4000 Å(2) ) is buried on trimer formation, which
also contributes to the resistance to deformability. The type I site
resembles that of cupredoxins, but with a more buried copper and a
similar number of ligand-orienting interactions. When the active-site
(type II) copper is absent, the histidines do not move, and a solvent
binds to His-135, His-100, and Asp-98. His-306 is likely protonated as
well as either His-100 or His-135. Asp-98, on the hydrophilic side of
the active site, has higher than average B-values in all of
the structures, although there are well localized solvent molecules
within hydrogen bonding distance of it. Nitrite replaces the copper
ligand solvent and binds asymmetrically with the oxygens toward the
copper, with Asp-98 in hydrogen bonding distance to the oxygen closest
to the copper.
For the one-electron reduction of
NO to NO and H
O, two proton
s
must be taken up. The most likely residues for providing these protons
are His-255 and Asp-98 and the water bound between them. Possible
hydrogen bonds extending from the copper water ligand are shown in Fig. Z2.
Figure Z2: Structure 2
Likely orientations for the water protons are shown, one toward
Asp-98 and the other toward the doubly protonated His-255. The plane of
His-255 is such that the N proton bond is directed
between O-1098 and OW-503. The hydrogen bond from the N
proton to O-279 and the weaker interaction between Thr-280
O
and His-255 N
prevent reorientation
of that histidine plane. Consistent with electron nucleus double
resonance experiments(15) , the two water ligand (position 503)
proton-copper distances would then be 2.85 and 2.7 Å.
When nitrite displaces the water, we suggest that one of the protons remains on Asp-98 so that effectively only OH is actually displaced, leaving the remaining proton required for the reaction to come from elsewhere. The role of His-255 might then be to provide a hydrogen bond to OW-1098, which also hydrogen bonds to Asp-98, in order to allow Asp-98 to temporarily hold a hydrogen on the other carboxylate oxygen, during the displacement reaction. Then, at pH values <5.5, Asp-98 would be protonated, disabling uptake of a proton from OW-503; at pH values >6.8, His-255 would be deprotonated, permitting a weak hydrogen bond from OW-503 and disabling the chain of proton transfers.
In addition to our electron density maps indicating that
NO binds to copper via its oxygens, a
model compound (22) also exhibits asymmetric binding of
NO
to the copper with Cu-O distances of 1.98 and 2.17
Å. The absence of an isotope effect when
NO
was replaced by
NO
in electron nuc
leus
double resonance studies (15) on the closely related A.
xylosoxidans NIR also supports oxygen rather than nitrogen binding
to copper. The EXAFS, EPR, and electron spin echo spectroscopy (24) study of a nitrite complex of a single copper hemocyanin
derivative favors a model in which nitrite binds to copper in the same
manner as the model complex above, but with a longer Cu-O bond.
The protein environment in the monocopper hemocyanin could cause the
decreased coupling seen in electron spin echo spectroscopy studies by
restricting the orientation of the three-atom
NO
, much as the protein environment in
NIR restricts NO
binding to
Cu
.
A model for binding of nitrite to NIR from A.
xylosoxidans based on EXAFS studies (27) is not consistent
with our results. Our study shows that the nitrite displaces the water
(Cu-OW) bound to the copper with the oxygens toward the
copper, but the EXAFS model is displaced from our x-ray results by the
vector between the two oxygens on the nitrite. Water 1098 found between
His-255 and Asp-98 with or without nitrite bound would prevent hydrogen
bonding of nitrite to His-255 as proposed in the EXAFS work. Ile-257
would also prevent nitrite from binding in the manner described. We do
not see an increase in Cu-N distances upon nitrite binding, as
their data suggest. Our Cu-S
distance is 2.56
Å, not 3.0 Å, as suggested by the EXAFS analysis, but this
could be a real difference between the ``green'' and
``blue'' NIRs.
We propose that
NO displaces the bound water, which
leaves as OH
, leaving a proton on Asp-98, and picks
up another proton as it exits along the hydrophilic side of the active
site. The Cu-ONO now has a redox potential appropriate for
electron transfer to it from the type I site. Reduction and the
protonated Asp-98 facilitate breakage of the NO bond, leaving one of
the NO
oxygens bound by the copper.
Cu
is regenerated, and NO leaves along the
hydrophobic side of the pocket. The Cu
ON
species must have a long enough lifetime to permit formation of
N
O or other nitrosylated products described by Hulse et
al.(30) . The protein may require absence of
nucleophiles, such as thiols, in this region since these would be
easily nitrosylated(35) .
The pH optimum of the reaction at
6.2, then, may result not from specific pH-dependent relocation of side
chains as anticipated, but from a delicate balance of the environment
at Asp-98 and His-255, required to make protons available to rebind to
the oxygen left on the copper. The high B-value or general
disorder associated with Asp-98 suggests it could act as a shuttle,
rotating about its C-C
bond to
bring protons to that site. Site-directed mutants of A. faecalis NIR are being made to test this idea.