Title: Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase
Abstract: Article1 November 2000free access Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase David M. Lawson Corresponding Author David M. Lawson Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK Search for more papers by this author Clare E.M. Stevenson Clare E.M. Stevenson Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK Search for more papers by this author Colin R. Andrew Colin R. Andrew Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, Beaverton, OR, 97006-8921 USA Search for more papers by this author Robert R. Eady Robert R. Eady Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK Search for more papers by this author David M. Lawson Corresponding Author David M. Lawson Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK Search for more papers by this author Clare E.M. Stevenson Clare E.M. Stevenson Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK Search for more papers by this author Colin R. Andrew Colin R. Andrew Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, Beaverton, OR, 97006-8921 USA Search for more papers by this author Robert R. Eady Robert R. Eady Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK Search for more papers by this author Author Information David M. Lawson 1, Clare E.M. Stevenson1, Colin R. Andrew2 and Robert R. Eady1 1Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK 2Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, Beaverton, OR, 97006-8921 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5661-5671https://doi.org/10.1093/emboj/19.21.5661 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Microbial cytochromes c′ contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 Å resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His–Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species. In contrast, CO binds 6-coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c′ are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain. Introduction Gas-sensing heme proteins play a central role in the regulation of important biological processes in mammals, plants and bacteria. Nitric oxide (NO)-, O2- and carbon monoxide (CO)-sensing systems have been identified (Rodgers, 1999; Hou et al., 2000). The regulation of gene expression by O2 in plant-associated nitrogen-fixing bacteria is well characterized and involves the heme protein FixL (Gong et al., 1998). CO regulates the transcription of enzymes that allow the photosynthetic microorganism Rhodospirillum rubrum to grow on CO as the sole energy source by binding to the heme protein CooA (He et al., 1996). NO plays a role in regulating a diverse range of physiological processes in higher organisms, such as the immune response to tumor cells, vasodilation and neuronal synaptic transmission (Denninger and Marletta, 1999). Many of these responses to NO are triggered by an increase in the level of the cellular second messenger cGMP. The latter is produced by soluble guanylate cyclase (sGC), which is activated by binding of NO to the heme domain. Particular interest surrounds the mechanism of activation and selective ligand recognition by these various sensor proteins. Cytochromes (cyt) c′ are found in the periplasmic space of a number of photosynthetic, nitrogen-fixing and denitrifying bacteria. In denitrifiers, they have been proposed to have a role in mediating NO transfer and protecting the organism from the potentially toxic levels of NO that may otherwise accumulate (Moir, 1999). The strong similarities in the distinctive reactivity and spectroscopic properties of the heme center in cyt c′ and that of sGC have been noted previously (Stone and Marletta, 1994). The ligand binding properties of both these proteins are anomalous when compared with other high spin hemoproteins since both sGC and cyt c′ form stable complexes with both NO and CO but not O2. In addition, it has been shown that NO forms 5-coordinate heme adducts with both sGC (Vogel et al., 1999) and cyt c′ (Iwasaki et al., 1991). In the absence of a crystal structure for any 5-coordinate NO–heme complex, the structural interpretation of spectroscopic data for NO binding to sGC is based solely on comparisons with the structures of 6-coordinate hemoprotein–ligand adducts and model complexes. Implicit in these comparisons is the assumption that NO binding is restricted to the distal side of the heme, a view that so dominates the interpretation of studies of ligand binding to hemoproteins that it allows the term 'distal dogma' to be used. The structural data for Alcaligenes xylosoxidans cytochrome c′ (Axcyt c′) we report here show that on binding NO, cleavage of the proximal Fe–His bond occurs and a 5-coordinate heme–NO complex is formed. However, contrary to expectations, we find NO to be bound on the proximal side of the heme. We also show that CO binding does not result in cleavage of the His–Fe heme bond and is ligated 6-coordinate on the distal side of the heme. These structures are the first for any gaseous ligands bound to cyt c′ and also the first for any 5-coordinate NO-ligated hemoprotein. Our findings that Axcyt c′ binds NO and CO to opposite sides of the heme center have clear implications for the mode of binding of NO and CO to sGC. Results and discussion Structure determination Axcyt c′ was isolated from the denitrifying bacterium A.xylosoxidans NCIMB 11015 essentially as described (Ambler, 1973). All the X-ray data presented here were collected under cryogenic conditions, where the unit cell typically shrinks by ∼5% relative to its volume at ambient temperature. Thus, for each structure, molecular replacement was used to place correctly the ambient temperature structure of the oxidized protein determined at 1.8 Å resolution (Dobbs et al., 1996) into the cryogenic coordinate frame prior to refinement and model building. In order to define potential structural changes to the protein and heme environment associated with the binding of ligands, we have determined the structure of the reduced and the CO- and NO-bound forms of Axcyt c′. However, since all X-ray data were collected at cryogenic temperatures, it was considered appropriate to re-determine the oxidized structure under these conditions as well. Axcyt c′ is a homodimer of 27.2 kDa with each subunit being comprised of an antiparallel four α-helix bundle containing a 5-coordinate c-type heme that is partially exposed to solvent (Figure 1). The subunits are arranged in a head-to-tail fashion, with both hemes on the same face of the dimer. Intersubunit contacts are mediated through residues in the A and B helices. The observations made by Shibata et al. (1998) place cyt c′ into two families according to the nature of the surfaces of these helices. Axcyt c′ belongs to Type 1, which have hydrophobic surfaces to their A and B helices and usually form globular, X-shaped dimers. It is notable, however, that the Chromatium vinosum protein will monomerize when CO is bound (Doyle et al., 1986), as will several others for which there are no structures (Ren et al., 1993). In contrast, the Type 2 proteins have A and B helices that are hydrophilic and they are either monomeric or form flattened dimers. Figure 1.Ribbon representation of a single subunit of the reduced Axcyt c′ structure showing the position of the heme. Also depicted are the side chains of the proximal His, Leu16 and the two Cys residues that form thioether bridges to the heme. The Leu blocks access to the vacant sixth coordination site in the distal pocket. The location of the crystallographic 2-fold axis is indicated, which is perpendicular to the plane of the paper. A 180° rotation of this subunit about the 2-fold axis generates the second subunit of the functional dimer. The structure is colored with respect to sequence number, starting with blue at the N-terminus and finishing with red at the C-terminus. This figure was produced using MOLSCRIPT (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997). Download figure Download PowerPoint The heme lies towards one end of the subunit and is essentially sandwiched between helices A and D, but also has interactions with helix C and the BC loop. Consistent with a body of spectroscopic data, it is 5-coordinate and covalently bound to the protein via two Cys–thioether bonds that are provided by a conserved Cys-X-X-Cys-His motif, where the His (residue 120) is the fifth ligand to the heme iron. This His lies in the proximal heme pocket, which is solvent exposed. In contrast, the distal pocket is buried and a hydrophobic residue, Leu16, blocks access to the vacant sixth coordination site of the heme. These are characteristics of Group 2 cyt c′, according to the distinctions made by Tahirov et al. (1996b), while Group 1 proteins have an aromatic residue in this position (Phe or Tyr) and the distal pocket is exposed to solvent (due to a channel between helices B and C). A more extensive description of the overall Axcyt c′ structure is not warranted here as this has been presented elsewhere (Dobbs et al., 1996). In this paper the focus will be on the heme environment in both the oxidized and reduced states, and the NO- and CO-bound forms. Oxidized and reduced structures In order to monitor potential redox-dependent changes in the heme environment, data were collected on both oxidized and reduced crystals of Axcyt c′ to resolutions of 2.05 and 1.90 Å, respectively. Overall, there was very little difference between the resultant models, having a root mean square (r.m.s.) deviation of 0.13 Å over all main chain atoms after least squares superposition. In both oxidation states, the proximal ligand, His120, is additionally hydrogen bonded through Nδ1 to an ordered water molecule that is accessible to bulk solvent. However, there was a clear difference in the position of the Arg124 side chain, which lies adjacent to the heme, being well defined in both structures. In the oxidized structure, the planar guanidinium moiety is perpendicular to the imidazole ring of His120 and almost parallel to the A pyrrole ring of the heme. In this position, the positive charge of the Arg presumably overlaps with the negative charge of the heme π system. In contrast, in the reduced structure this side chain is parallel to the His (Figure 2). Clearly, this residue senses the oxidation state of the iron, although the mechanism is not apparent. The possibility that the oxidized Axcyt c′ crystal may have been photo-reduced during X-ray data collection has been considered, even though the experiment was performed at 100 K. However, this cannot have occurred to any significant extent because of the unambiguous differences described above. At the resolutions of these two structures, the observed difference of 0.1 Å in the His Nϵ2–Fe bond lengths, which are 2.0 and 2.1 Å, respectively, in the oxidized and reduced models, may not be significant. In both structures the heme group is distinctly puckered and the Fe is displaced out of the heme plane by ∼0.3 Å towards the proximal His. This is consistent with the Fe remaining high spin in both structures, as would be expected given that the crystals were grown under alkaline conditions. Moreover, the sixth coordination site remains vacant in both models. Figure 2.Stereo diagrams of the final 2mFobs – dFcalc electron density maps contoured at 1σ for the (A) oxidized, (B) reduced, (C) NO-bound and (D) CO-bound Axcyt c′ structures. The atoms are colored as follows: Fe, green; S, yellow; O, red; N, blue; C, gray. The red crosses indicate water molecules. In this view, both the left-hand and rear edges of the heme are accessible to solvent, as is evident from the presence of water molecules in these regions. In the interests of clarity, the thioether bridges to the heme group, which would be in the foreground, have been omitted. In the oxidized and NO-bound structures in particular, there is a large unassigned region of electron density within hydrogen bonding distance of the side chain of Arg124. Attempts to model buffer and cryoprotectant components here were not convincing. However, a reasonable fit was obtained with a sulfate anion (present in the precipitant), but this gave high temperature factors after refinement. (E) A least squares superposition of all four structures based on all main chain atoms. They are colored as follows: oxidized, yellow; reduced, red; NO-bound, green; CO-bound, blue. In the interests of clarity, all bonds to the heme Fe have been omitted. All images were produced using the programs O and OPLOT (Jones et al., 1991). Download figure Download PowerPoint NO-bound structure We originally determined the NO-bound structure using X-ray data to 1.95 Å resolution (not shown). The resultant electron density maps clearly indicated the binding of NO as a 5-coordinate adduct. This is consistent with the interpretation of earlier spectroscopic studies, which indicated that at physiological pH values the His–Fe bond is readily cleaved on NO binding (Yoshimura et al., 1986). However, unexpectedly, binding occurs on the proximal face of the heme with the concomitant displacement of His120, mainly through a 111° rotation about the Cα–Cβ bond. Unfortunately, at this resolution the absolute conformation of the ligand could not be modeled confidently. In order to resolve this issue, data were collected to near atomic resolution. In electron density maps subsequently calculated at 1.35 Å resolution, it was possible to model NO binding in two alternative bent conformations, each with half occupancy, with Fe–N–O angles of 124 and 132° for the two conformers and an average Fe–NO bond length of 2.0 Å (Figure 2). In one binding mode, the NO has no specific interactions with the protein (NO 1), whilst in the other a hydrogen bond forms with Nη1 of Arg124 (NO 2). This interaction is possible because the Arg side chain has adopted a conformation similar to that observed in the oxidized structure, where it stacks against the heme plane. It is notable that the side chain of His120 in this structure is less well defined in the electron density, presumably because it has lost the covalent bond to the iron, instead having only a single hydrogen bond to a solvent molecule through atom Nϵ2. After refinement, the two alternative conformations for NO had essentially equivalent thermal parameters (Table I; Figure 3), suggesting that the assignment of half occupancy to each was valid. This is surprising since conformation 2 would appear to be the more favorable because it has a hydrogen bonding interaction with the protein, whilst conformation 1 does not. Figure 3.Omit difference maps for the (A) NO- and (B) CO-bound Axcyt c′ structures contoured at 3.5σ. All the displayed atoms were omitted from the refinements (see Materials and methods). However, Arg124 was retained in the refinement, in order to remove distracting density, in particular from the NO ligand. The view is chosen to emphasize the flattening of the heme plane upon binding of CO. The atoms are colored according to their temperature factors, which all lie roughly in the range 10–40 Å2, where dark blue indicates a low value increasing through light blue, dark green, light green, yellow and orange, to red, which indicates a high value. Note that the side chain of His120 in the NO-bound structure and the side chain of Leu16 and the propionate group A in the CO-bound structure are less well defined in the electron density and have relatively high thermal parameters. See main text for a further explanation. Download figure Download PowerPoint Table 1. Summary of X-ray data and model parameters for A.xylosoxidans cytochrome c′ Oxidized Reduced NO-bound CO-bound Data collectiona site PX7.2 (SRS) ID14-2 (ESRF) ID14-2 (ESRF) BM30 (ESRF) detector Mar300 image plate ADSC Quantum-4 CCD ADSC Quantum-4 CCD Mar345 image plate wavelength (Å) 1.488 0.933 0.933 0.979 cell parameters: a = b, c (Å) 52.9, 182.3 52.8, 182.6 53.0, 180.9 53.6, 180.9 resolution range (Å) 40.0–2.05 40.0–1.90 40.0–1.35 40.0–1.95 unique reflections 10 205 12 387 33 925 12 158 completeness (%) 99.0 (98.9) 97.1 (84.5) 98.8 (93.1) 98.7 (86.8) redundancy 8.1 4.9 6.8 9.9 Rmergeb 0.109 (0.349) 0.041 (0.122) 0.039 (0.227) 0.066 (0.310) <I>/<σI> 18.8 (5.2) 31.6 (8.3) 39.8 (3.9) 33.2 (5.8) Refinement Rcrystc (based on 95% of data) (%) 19.2 21.7 19.4 20.3 Rfreec (based on 5% of data) (%) 25.2 27.3 22.0 25.8 DPId (based on Rfree) (Å) 0.18 0.16 0.06 0.16 residues with most favored φ/ψe (%) 93.6 94.5 94.4 95.4 r.m.s. deviation bond distances (Å) 0.017 0.016 0.011 0.016 r.m.s. deviation angle distances (Å) 0.037 0.033 0.024 0.034 Average temperature factors (Å2) main chain atoms 20 19 14 20 side chain atoms 23 21 17 23 heme 17 15 13 18 gaseous ligandsf (1/2) – – 16/17 27/34 overall 23 22 18 23 R.m.s. deviation versus reduced structureg (Å) 0.13 – 0.15 0.60 a aThe figures in parentheses indicate the values for the outer resolution shell. b bRmerge = ∑(|Ij – <Ij> |)/∑<Ij>, where Ij is the intensity of an observation of reflection j and <Ij> is the average intensity for reflection j. c cThe R factors Rcryst and Rfree were calculated as follows: R = ∑(|Fobs – Fcalc|)/∑|Fobs| × 100, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. d dDiffraction component precision index (Cruickshank, 1999), an estimate of the overall coordinate errors calculated in REFMAC. e eAs calculated using PROCHECK (Laskowski et al., 1993). f fSee Figure 3. Note, the two NO molecules have half occupancy whilst the two CO molecules have unit occupancy. g gAfter least squares superposition based on all main chain atoms. CO-bound structure In the 1.95 Å resolution structure of the CO-bound adduct, the heme is 6-coordinate with the CO bound to the distal face of the reduced heme. The CO binds in an almost linear configuration with an Fe–C–O angle of 167° and Fe–CO bond length of 2.1 Å. This binding induces a cascade of conformational changes. The side chain of Leu16, which mediates access to the distal heme face, is displaced to one side, largely by a simple 134° rotation about the Cα–Cβ bond. In doing so, it pushes pyrrole ring A of the heme group upward into the proximal pocket (maximum displacement ∼1 Å). This movement effectively flattens the heme group. The positions of the Fe and the proximal His also move in the same direction, but to a lesser extent, with the net effect of restoring the Fe into the heme plane. The conformation adopted by Arg124 is similar to that found in the reduced structure, although it is slightly higher due to the upward movement of the heme group (Figure 2). The side chain of Leu16, the propionate group A of the heme and the CO molecule all have relatively high thermal parameters compared with the overall value for the heme (Figure 3). Indeed, in maps calculated from a previous data set (not shown) there was electron density for Leu16 in both the conformation seen here and in the conformation observed in the other three structures overlapping with density for CO. Thus, the elevated thermal parameters of the CO may indicate that it is not fully occupied here as well. During the process of model building, an elongated peak of electron density was observed adjacent to and within hydrogen bonding distance of Nδ1 of the proximal His residue. The single water molecule observed in both the reduced and oxidized structures (having temperature factors of 27 and 31 Å2, respectively) was insufficient to account for all the electron density. However, the inclusion of a second CO molecule at this position gave a good fit. After refinement at full occupancy, the CO had temperature factors of 37 and 31 Å2 for the C and O atoms, respectively, as compared with the value of 18 Å2 for the Nδ1 atom of His120, with a hydrogen bond length of 2.9 Å. This interaction with CO can be likened to that seen in many hemoproteins where the proximal His forms a hydrogen bond with either a main chain carbonyl or a side chain carboxyl group (Poulos and Kraut, 1980; Harutyunyan et al., 1996). The presence of this additional CO shows that, at least in this structure, the proximal His must be protonated. The possibility that this CO has arisen from X-ray-induced photo-dissociation of heme-bound CO during data collection cannot be excluded and may provide an explanation for the reduced occupancy of the CO bound to heme. Recent experiments with carbonmonoxymyoglobin crystals have shown that laser irradiation induces photolysis of the Fe–CO bond, resulting in CO becoming trapped in alternative positions in both the proximal and distal pockets (Brunori et al., 2000; Chu et al., 2000; Ostermann et al., 2000). In these cases, the CO migrates to specific sites but does not hydrogen bond to the protein. Of the two ligand-bound Axcyt c′ structures presented here, the CO-bound form differs the most with respect to the reduced structure after least squares superposition (Table I). The overall r.m.s. deviations were 0.15 and 0.60 Å for the NO- and CO-bound models, respectively. CO binding in other cyt c′ has been shown to induce dimer dissociation (Ren et al., 1993). This has not been reported for Axcyt c′, although these differences could indicate that such a dissociation might occur in this protein outside the constraints of the crystal lattice. However, the differences did not exceed 1.2 Å for main chain atoms and were largely restricted to the N-terminus and the AB, BC and CD loops, whilst the dimer interface remained virtually unchanged. The ligand-bound structures presented here are not the first for a cyt c′. The structure of reduced Rhodobacter capsulatus cyt c′ (Rccyt c′) with n-butylisocyanide (BIC) bound to the heme has been determined at 2.4 Å resolution (Tahirov et al., 1996a). This structure has much in common with the CO-bound Axcyt c′ structure. First, the heme is 6-coordinate, with the ligand binding in the distal pocket with an average Fe–C–N angle of 165° (there are two subunits per asymmetric unit). This binding displaces Phe14 (equivalent to Leu16 in Axcyt c′), which mediates access to the sixth coordination site. Furthermore, this movement pushes pyrrole ring A of the heme upward into the proximal pocket and the Fe moves back into the porphyrin plane. In this BIC-bound structure, Arg126 (equivalent to Arg124 in Axcyt c′) is parallel to the plane of the imidazole ring of the proximal His and perpendicular to the heme plane. This contrasts with its position in the ligand-free oxidized Rccyt c′ structure, where it stacks against the heme plane (Tahirov et al., 1996b), as it does in Axcyt c′. The authors inferred that this side chain flipping is attributable to the effects of BIC binding (Tahirov et al., 1996a), whereas our Axcyt c′ structures indicate that this is simply the result of the transition from an oxidized to a reduced state. The side chain is merely shifted upward by <1 Å upon CO binding to the ferrous heme of Axcyt c′ (Figure 2). The effects of BIC binding in Rccyt c′ are more widespread than those of CO binding in Axcyt c′, possibly because a larger ligand is involved. In particular, at least three other residues in the distal pocket become re-oriented and propionate group A flips around such that it resides on the proximal side of the heme (Tahirov et al., 1996a). This results in the loss of a water molecule that was hydrogen bonded between the two propionate groups in the oxidized Rccyt c′ structure. This water is structurally conserved in several other cyt c′ structures and has been suggested to play an important functional role (Tahirov et al., 1996a). In contrast, this water is retained in all four Axcyt c′ structures reported here. Steric constraints within the distal heme pocket most likely favor linearly bound adducts such as CO and discriminate against those ligands that prefer a bent configuration, like O2 and NO. O2 binding needs the proximal histidine as a strong electron donor and therefore can only bind to the distal side in 6-coordinate heme complexes. However, the steric hindrence, possibly involving Leu16, may prevent this in Axcyt c′. These constraints could also help to explain the transient nature of the 6-coordinate NO species of Axcyt c′ discussed below. Structural comparison of the mode of NO binding with other hemoproteins 5-coordinated nitrosyl heme was first discovered in a spectroscopic study of the α-hemes of human deoxyhemoglobin (Szabo and Perutz, 1976). However, previous crystallographic studies of the NO complexes of hemoproteins are restricted to 6-coordinate adducts with the proximal histidine still ligated to the Fe atom. In addition to this mode of binding, in hemoglobin NO has been reported to bind to a Cys residue (β93) outside the heme pocket, to form an S-nitroso adduct (Chan et al., 1998), and is therefore not directly relevant to this discussion. This modification cannot occur in Axcyt c′ as there are no free Cys residues. Significantly, none of these structures has revealed the binding of NO to the proximal face of the heme that we find for Axcyt c′. The first reported nitrosyl hemoprotein structure was that of ferrous horse hemoglobin (Deatheridge and Moffat, 1979) to a resolution of 2.8 Å. In this structure the ligand was modeled into a difference electron density map with an Fe–NO bond length of 1.7 Å and Fe–N–O angle of 145°, but was not refined. More recently, a ferrous nitrosyl leghemoglobin structure refined at 1.7 Å resolution gave similar values of 1.7 Å and 147°, respectively, for the Fe–NO bond length and the Fe–N–O angle (Harutyunyan et al., 1996), whilst average values of 1.8 Å and 127° were obtained for the Fe–NO bond length and the Fe–N–O angle, respectively, from a 1.8 Å resolution ferrous nitrosyl hemoglobin structure (Chan et al., 1998). In contrast, a 1.7 Å resolution structure of ferrous nitrosyl myoglobin (Brucker et al., 1998) showed a very acute Fe–N–O angle of 112° with an Fe–NO distance of 1.9 Å. In the latter study, the authors postulated that this acute angle may have been influenced by the strength of the proximal bond, hydrogen bonding interactions between the ligand and the distal histidine and, possibly, crystal packing forces. Nitrophorins are a class of NO transport proteins found in the saliva of blood-feeding insects and function as vasodilators and anticoagulants (Weichsel et al., 2000). The Fe is 5-coordinate with a His residue as the proximal ligand and spectroscopic studies indicate that both the ferric and ferrous oxidation states of nitrophorins bind NO. However, the ferric form of the heme in these proteins is stabilized in some way to prevent autoreduction by NO, allowing the freely reversible binding of NO from this 6-coordinate species. The crystal structure of the NO- complexed form of nitrophorin 1 has been determined at 2.3 Å resolution (Ding et al., 1999). The structure contains two molecules per asymmetric unit and shows the Fe–NO moiety to be bent by an average of 130° with an Fe–NO bond length of 2.0 Å. Although formally ferric, since the X-ray data were collected at ambient temperature, it is possible that the heme Fe may have been photo-reduced during the experiment, thereby accounting for the acuteness of the Fe–N–O angle. Very recently, a 1.4 Å resolution crystal structure of the 6-coordinate NO adduct of nitrophorin 4 was presented in which the NO was discretely disordered (Weichsel et al., 2000). In one conformation, the Fe–N–O bond angle was 177° with an Fe–NO bond length of 1.5 Å, whilst in the other the Fe–N–O bond angle was 110° with an Fe–NO bond length of 2.6 Å. Rather than a mixture of ferric and ferrous states of the heme Fe, the authors interpreted this as a mixture of bound and unbound or loosely ligated NO molecules. The cd1 heme-containing nitrite reductase of Thiosphaera pantoteopha forms NO as a product of nitrite reduction and has been studied by time-resolved crystallography. The catalytic site is a 6-coordinate d1 heme with axial ligands His and Tyr in the resting state. During catalysis, the Tyr residue is displaced on reduction of the heme by internal electron transfer from the c heme. Nitrite is N-bonded to the reduced d1 heme and is reduced to NO, which has an Fe–N–O bond angle of 131° and an Fe–NO bond length of 2.0 Å in a structure refined at 1.8 Å resolution (Williams et al., 1997). Spectroscopic measurements of the nitrite reductase crystals indicated that the heme–NO-bound product is in the ferrous oxidation state. In structural studies on the same enzyme from Pseudomonas aeruginosa, the d1 heme in the resting enzyme was not directly ligated by a Tyr, but is linked to one via a hydroxide ion (Nurizzo et al., 1998). In the structure of the NO-bound form of this enzym