©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Structure of Copper-nitrite Reductase from Achromobacter cycloclastes at Five pH Values, with NO Bound and with Type II Copper Depleted (*)

(Received for publication, May 18, 1995; and in revised form, August 23, 1995)

Elinor T. Adman (§) J. W. Godden Stewart Turley

From the Department of Biological Structure, University of Washington, Seattle, Washington 98195-7420

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(2) is the ultimate produc t. This step requires donation of electrons to NIR (^1)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 beta-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 (^2)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(2) 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(2). 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(2) complex with NIR. An extensive structural description is provided for future site-directed mutagenesis studies and other anticipated comparisons.


EXPERIMENTAL PROCEDURES

Crystal Growth

Cubic crystals of A. cycloclastes NIR, prepared as described previously(23) , can be grown at pH 5.0 in 20 mM potassium phosphate, 33% saturated ammonium sulfate. They tolerate pH adjustment with NaOH up to pH 8.0. At higher pH values, both orthorhombic and cubic forms are found, whereas at the lower pH values, only the cubic form is found. Cubic crystals can be transferred to, or grown in, acetate buffer (usually 100 mM).

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). (^3)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.

Data Collection

Diffraction data were collected at ambient room temperature on a Siemens X-100 area detector on a Rigaku RU200 rotating anode with a graphite monochromator. The power was set typically at 50 kV and 60 mA to record <1,000,000 total counts on the detector. The detector to crystal distance was usually 12-14 cm, and no helium paths were employed. The recorded data were processed using XENGEN(25) . The T2D data set was collected on an Raxis image plate system at Molecular Structure Corp. These data were processed by the software package from Molecular Structure Corp.(26) . Statistics on data collection are summarized in Table 1. Table 2summarizes the scaling parameters and agreement between data sets.





Structure Solution, Model Building, and Refinement

The programs X-PLOR (versions 3.0-3.2; A. T. Brunger, Yale University, New Haven, CT) and O (version 5.8.2 for an Evans & Sutherland ESV) (28) were used for refinement and map fitting. MOLSCRIPT (29) and XTALVIEW (version 2.0; Duncan E. McRee, San Diego Super Computer, San Diego, CA) were used for preparing Fig. 4and Fig. 6and Fig. 7Fig. 8Fig. 9, respectively. CCP4 (31) was used for most other calculations. The parameter files of Engh and Huber (32) were used for the energy terms in refinement. Copper energy parameters were accorded zero weight in the refinement through ``constraint interaction'' weights. The definition of the type I site is straightforward, but that for the type II site depends on which crystal form is being used. In the cubic crystal form, the type II copper is covalently bonded to residues from each of two adjacent symmetry-related molecules. The most straightforward way to deal with it in X-PLOR is to make sure that all interactions between His-306 N with the copper and its ligand OW are set to zero weight. In all forms except for T2D and NT1, a water is treated as a fourth ligand to the type II copper, but no restraints were used for it. The water found in the T2D structure is designated ``T2D-OW,'' while that in the other structures is ``Cu(B)-OW.''


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(B)-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(B) 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(B) 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 - Fmaps for T2D (a), PH5 (KP and HR) (b), and NT1 (c and d). In each, T2D-OW, Cu(B)-OW, or NO(2) 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(B)-OW. It can be seen that in a, T2D-OW is closer to Cu(B) than Cu(B)-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.

Orthorhombic Form

The Patterson correlation methods in X-PLOR were used to solve the orthorhombic structure, using a trimer generated from the current model of the cubic crystal form. The model was put in a 120-Å box, and maximum vector lengths of 30 Å and data from 20 to 6 Å were used. The rotation found from a separate Crowther fast rotation function calculation was intermediate on the list of rotation function peaks from X-PLOR; however, rigid body refinement of first the trimer and then each monomer definitely selected this peak (correlation coefficient of 0.178 versus 0.03 for others). The translation function maximum yielded a model that refined from R = 0.45 to 0.34 as a rigid trimer and to 0.31 as rigid monomers for 20 to 4-Å data. Other models with translation function values near the maximum did not refine. Examination of the packing interactions showed that residues 13 and 14 of monomer A overlapped residues 336-340 of monomer A in a neighboring trimer; new positions for these residues were visible in difference maps.

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.

Cubic Forms

About 20 rounds of positional and B refinement and map checking were carried out with the T2D data, until the main peaks remaining in the difference maps were judged to be additional low occupancy solvent. Good density for the first eight residues is completely lacking. A model near the end of the T2D refinement was used for the other refinements when it became apparent that the model was improved over those being done independently. Individual solvent models were retained for each, and alternate conformations were retained for the HR model. The KP and PH5 models were derived from the HR model. No less than three rounds of examining difference maps and incorporating solvent were done for other models (in some cases, more). An additional simulated annealing run of the HR model without solvent but with alternate side chains was performed, and that resultant model passed once more through a positional run and a B-factor run. The appearance of the Luzatti plot for this suggested that it was not yet completely converged. However, the coordinates did not significantly differ, so further refinement was not pursued. Final R-factors and refinement statistics are summarized in Table 3and Table 4. ( Table 5summarizes the agreement between each of the models with its own data and with data not used in its refinement. Table 6gives the root mean square deviation between coordinate sets.) Fig. 2shows R-value versus resolution for each of the coordinate sets, and Fig. 3shows average B-value versus residue number for final T2D, PH5, 62, and NT1 models. KP and HR are nearly identical to PH5, and 68 to 62. Fig. 4is a plot of two views of the fold of a monomer, with residues defining secondary structure labeled. The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY (codes are 2NRD for the PH5.4 (HR) set, which is a replacement for the 1NRD alpha-carbon coordinate set, and 1NIA (62), 1NIB (68), 1NIC (KP), 1NID (NT1), 1NIE (PH5), and 1NIF (T2D) for the other sets).










Figure 2: R-factor versus resolution (expressed as sin^2/^2) 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.




RESULTS

Errors, Refinement, and General Geometry

All of the models agree with all of the data nearly equally well as shown by Table 5and Table 6. The apparently worse agreement seen for all of the models versus T2D data is attributable to higher R-factors in the shells of data between 1.6 and 2.0 Å. The only significant differences in the structures are at the type II copper site. Although some cycles of positional and B refinement were done for the KP, PH5, and HR structures, they clearly are strongly correlated with the T2D structure. Calculation of displacement parameter probability weighted difference distances (^4)are on the order of 0.04-0.09 Å among the various structures, the smaller values clearly indicating high correlation, but also that the errors may be less than the overall 0.18 Å suggested from the Luzatti plot (Fig. 2).

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 beta-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 beta-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 beta-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.

Alternate Conformations, cis-Prolines, and Other Remarkable Conformations

There are only two cis-prolines in the structure, residues 23 and 69, both involved in interdomain contacts. Pro-69 is part of the ``dogleg'' stretch (described below), while Pro-23 is at the turn at the top of the N-terminal loop, a loop that is ``extra'' with respect to the cupredoxin fold. Very few unusual side chain conformations are detected except for His-306, the copper ligand from the second molecule. Glu-165, Asn-221, Gln-247, His-306, Phe-320, Leu-330, and Ser-339 all have mean deviations >30° from usual (1)-values. Each is involved in slightly different (but reasonable) local side chain packing interactions (except for Ser-339, which is just not well fit). Val-133 also has an deviation >12°, probably the result of special interactions with copper ligands, for its carbonyl oxygen is hydrogen-bonded to His-100 nitrogen, and His-100 is a copper ligand.

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 beta-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 beta-strand and is exposed, but other valines are also exposed and not distinctly disordered. No residues interior to the beta-sheets are disordered.

Protein Fold and Comparison to Cupredoxins

Fig. 4and Fig. 5illustrate the topology of each domain and summarize the hydrogen bonding within the monomer, between monomers, and with solvent. ( Table 7annotates all of the hydrogen bonds.) Each of these domains contains the core topology of the cupredoxins (collective name for plastocyanin, azurin, pseudoazurin, etc)(34) . This consists of an N-terminal sheet with four strands (two pairs of antiparallel strands (beta2,beta1,beta4,beta7 and beta11,beta10,beta13,beta17) joined at the center by parallel strands (beta1,beta4 and beta10,beta13)) and a C-terminal sheet, also of four strands (a pair of antiparallel and a pair of parallel strands (beta5,beta8 and beta9,beta3; beta14,beta18 and beta19,beta12) joined at the center by antiparallel strands (beta8,beta9 and beta18,beta19)). So-called doglegs (34) connect the two sheets between beta2 and beta3 and between beta11 and beta12 in ea ch domain. The side of the barrel opposite the dogleg is the most variable among cupredoxins and is also variable here. In domain 1, a single strand (beta6) of residues extends the C-terminal sheet with a pair of hydrogen bonds near the top while filling in the rest of the space between the sheets with somewhat irregular geometry. Strand beta7 (residues 117-126) hydrogen bonds in an antiparallel fashion with the C-terminal tail, an important interaction in defining the active-site entrance. In domain 2, the connecting residues extend both of the sheets: beta16 adding to the C-terminal sheet in the same manner as beta6 in domain 1 and beta15 adding to the N-terminal sheet. This loop (residues 263-280) is involved in extensive subunit interactions in the trimer, including formation of the ``back wall'' of the active site. Last, it can be seen that the Greek key beta-barrel of domain 1 of NIR has two strands formed from N-terminal residues 9-35 (the front two strands in the left-most domain in Fig. 4a) that are extra with respect to the cupredoxins and that loosely extend each of the beta-sheets. They have an extended conformation: residues 13-18 hydrogen bond to residues 38-40. Residues 24-30 make no direct hydrogen bonds with beta3, but make one water-mediated interaction.


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 beta-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 beta10 and beta11 and provides interdomain interactions with the topologically analogous loop connecting beta1 and beta2 in the first domain described earlier. The only other helical regions are alpha(2), which provides three ligands to the type I copper in domain 1, and its topological counterpart in domain 2, alpha(3), 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(n) loop (Tyr-301 OH to Gln-297 nitrogen and oxygen), common to most Greek-key beta-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 beta-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.

Interdomain, Subunit, and Solvent Interactions

The interdomain surface is largely hydrophobic. Unlike the interaction between the Greek-key beta-barrel domains of immunoglobulins that form another beta-barrel between them, convex sides of the beta-sheets appose each oth er: the convex side of the C-terminal sheet of domain 1 is closest to the convex side of the N-terminal sheet of domain 2. The two beta-barrels are associated with each other such that viewed roughly along the normal to the first beta-sheet, the next sheet is rotated -10° about its normal, the next another -30°, and the fourth another -10°. The main axes of the two barrels are rotated 35° from parallel. Smaller residues are found at the center of the interdomain interaction, while larger residues fill in as the sheets diverge. Leu-72, Val-74, Ala-153, Met-155, Leu-157, Val-133, and Val-131 are at the center of the domain 1 C-terminal sheet, while Ala-288, Ala-290, Val-244, Leu-242, Tyr-177, and Ile-175 are at the center of the domain 2 N-terminal sheet. Of these 13 residues, six are conserved and six semiconserved. Tyr-177 is an exception, reported as Thr in P. aureofaciens NIR(10) .

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^2 and another near 45 Å^2. All of the solvent molecules with B <25 Å^2 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.

Global Differences between Different Forms

The most significant differences seen among the seven structures occur at the type II copper. Systematic differences such as shifts of entire beta-sheets were not seen in difference maps. When a trimer from the cubic crystal form is superposed with the trimers in 62 and 68 by generating the transformation matrix from a single monomer, there appears to be a small residual rotation of the remaining two subunits. While the difference is visible on the graphics, most, if not all, local interactions are maintained as well as all Cu-Cu distances in the trimer. (Cu(A)-Cu(B) is 13 Å within a monomer. Between monomers, the distances are as follows: Cu(A)-Cu(A), 29.7 Å; and Cu(B)-Cu(B), 43.7 Å. The two other Cu(A)-Cu(B) distances are 35.5 and 39.9 Å.)

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.

Geometry and Environment of Copper Sites

Type I Copper

The mean values of the copper-ligand bond lengths in the type I site (Table 8) fall slightly to the outside of the range of distances tabulated by Libeu et al.(^5)for pseudoazurin and plastocyanin, with distances to His-95, His-145, and Met-150 all shorter than their counterparts in the cupredoxins and to Cys-136 slightly longer. The most obvious difference is the copper-Met distance of 2.56 Å, markedly shorter than the range of 2.67-2.83 Å seen in the oxidized forms of the cupredoxins cited above.



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 alpha(1) 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.^2 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.

Type II Copper Site, T2D Water, Copper Water Ligand, and Nitrite Binding

Difference maps calculated using refined T2D model phases and various pairs of data sets clearly show that there is variable occupancy of the copper at the type II site ( Fig. 7(a-d) and 8 (a-d)) and that this is the only significant identifiable difference among the various data sets. When all of the difference maps are put on a common scale, the relative heights suggest that the ordering of the copper occupancy is HR > NT1 > KP PH5 ( Fig. 7and Fig. 8). These differences do not correlate with pH. Fig. 7also shows differences in the thermal parameters of Asp-98: both pH5.0 and pH6.0 (KP) have a more ordered Asp-98 than pH5.4 or NT1. Fig. 7and Fig. 8also show that there are changes at the ligand Cu(B)-OW site. In contrast to the type II site, the type I site shows consistent occupancy throughout the data sets (data not shown).

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(o) - F(c) difference maps for each of the data sets, with the water ligand not included in the F(c) 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(2)O distances in small compounds (1.96-2.41 Å in CuSO(4)bullet5H(2)O and 2.0 Å in CuH(2)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(B) water is not perfectly tetrahedrally disposed, but quite close to it.



Active-site Pocket

The type II copper and its ligands are situated in a pocket formed by apposition of domain 2 of one monomer and domain 1 of another, as shown in Fig. 10(a and b) (loosely referred to as the ``clown face'' orientation, with the type II copper at the nose and the extended C-terminal arm forming the lower part of a ``smile''). This pocket can be visualized in the following way: curl the fingers of each hand, keeping thumbs aligned with the forefingers, and bring the palms together at the heel so that the curled knuckles now touch. Then, keeping knuckles together, rotate the right hand slightly back. The curled fingers represent the small helical loop between the C-terminal strands of each beta-barrel (or the ``eyes'' of the clown), while the back and palms of the hands represent the N- and C-terminal beta-sheet of each domain. The pocket is formed because residues protruding from the helical loop do not allow residues just under the loop to get very close to one another. The residues under the loop provide ligands to the copper as well as residues, which line the pocket, that play a role in binding and releasing substrate and product.


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(B). 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^2). 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 beta-sheet. Its 2-fold counterpart, residues 255-260 (HLIGGH), is also conserved and forms an irregularity on the edge of its beta-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.


DISCUSSION

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^2 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(2) to NO and H(2)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(2) binds to copper via its oxygens, a model compound (22) also exhibits asymmetric binding of NO(2) to the copper with Cu-O distances of 1.98 and 2.17 Å. The absence of an isotope effect when ^14NO(2) was replaced by NO(2) 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(2), much as the protein environment in NIR restricts NO(2) binding to Cu(B).

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(B)-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(2) 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(2) oxygens bound by the copper. Cu is regenerated, and NO leaves along the hydrophobic side of the pocket. The CuON species must have a long enough lifetime to permit formation of N(2)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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 31770. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biological Structure, P. O. Box 357420, University of Washington, Seattle, WA 98195-7420. Tel.: 206-543-6589; Fax: 206-543-1524.

(^1)
The abbreviations used are: NIR, nitrite reductase; EXAFS, extended x-ray absorption fine structure. Data and coordinate sets from crystals at pH 5.0, 5.4, 6.0, 6.2, and 6.8, type II copper-depleted, and nitrite-soaked are designated PH5, HR, KP, 62, 68, T2D, and NT1, respectively (HR and KP are mnemonics for ``high resolution'' and ``potassium phosphate''). The copper in the type I site is designated Cu(A), and that in the type II site, Cu(B). No relationship to those sites in cytochrome oxidase is intended.

(^2)
Murphy, M. E. P., Turley, S., Kukimoto, M., Nishiyama, M., Horinouchi, J., Sasaki, H., Tanokura, M., and Adman, E. T.(1995) Biochemistry34, 12107-12117.

(^3)
B. Averill and Y. Wang, personal communication.

(^4)
C. Peters-Libeu and E. T. Adman, manuscript in preparation (presented at the American Crystallographic Association Annual Meeting, July 23-28, 1995, Montreal).

(^5)
C. Peters-Libeu, S. Turley, and E. T. Adman, manuscript in preparation.


ACKNOWLEDGEMENTS

A. cycloclastes NIR has been very generously provided by J. LeGall and M.-Y. Liu. Bruce Averill and Yaning Wang provided T2D material. Discussion with Michael E. P. Murphy during the course of the work, critical reading of the manuscript by Ron Stenkamp, and help from Myrna Jewett and Karen Galt on preparation of the figures are all gratefully acknowledged. The gracious hospitality of colleagues at the University of Tokyo, where this manuscript was partially written, is much appreciated.


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