Irrespective of the bacterial species from which they are isolated these enzymes have the same heme/nonheme iron active site that catalyzes N—N bond formation in the reaction ofEquation (5). However, some nitric oxide reductases (NORs) exhibit a different subunit composition and type of center involved in electron transfer to the active site. The prototype bacterial NOR, isolated from Gram-negative organisms P. stuzeri, Pa. halodenitrifcans, and P. denitrificans, is dimeric with the subunits encoded by norBC.5,6,88–90 These are membrane-bound enzymes con- taining both nonheme and heme iron, and despite their sequence homology with heme copper oxidases, prototype bacterial NOR does not contain copper. No structure of the bacterial NORs has been determined so the homology modeling and spectroscopy provide the basis for the nature and organization of the redox centers these enzymes contain:
2NO + 2e + 2H– + N O + H O [ '2 2 Eo(pH 7.0) = +1.18 V] ð5ị
The dimeric NOR from Bacillus azotoformans contains a CuA center located in the smaller subunit, in place of thec-type heme of prototype NOR. Menahydroquinone, but not cytochrome c, functions as electron donor.91
Asecond variant, the enzyme from Ralstonia eutropha H16 (previously Alcaligenes eutrophus H16), is monomeric (encoded by norB) and lacks the small subunit that binds the electron donating centers of the other enzymes.92 Residues proposed to ligate hemes b and FeB are conserved in norB of R. eutropha and the enzyme contains heme b and nonheme Fe in a 2:1 ratio. The enzyme has an extended N-terminus of 280 amino acids that is essential for activity, with a proposed role in allowing quinols rather than cytochromecto function as electron donor.
These NORs, which are also membrane bound, have biochemical and spectral properties indicat- ing that the catalytic center is the same as in prototype NOR.
8.28.4.1 Prototype NOR Containing Heme c, Heme b, and Nonheme Fe
Although NOR may be associated with additional subunitsin vivo,3fully functional NOR prepar- ations isolated following detergent treatment of membranes are dimeric. The smaller subunit (NorC, 17 kD) is predicted to have a single transmembrane helix at the N terminus that anchors the periplasmic C terminus to the membrane. This subunit has a single Cys–Xaa–Xaa–Cys–His hemecbinding motif. The larger subunit (NorB, 53 kD) is predicted to have 12 transmembrane helices and carries the low-spin bis-His coordinated hemeband the dinuclear hemeb3–FeBcenter.
DNAsequence comparisons of NorB with subunit I of CcO show that NOR is a divergent member of the superfamily of the respiratory heme/copper oxidases. The structure of CcO shows the active site to be a high-spin heme located5 A˚ from a copper ion (CuB) forming a magnetically interacting dinuclear center.93 As described below, the analogous center of prototype NORs forming the catalytic center is a high-spin b-type heme (hemeb3) and a nonheme iron center (FeB).
Prototype NORs have been solubilized and purified from a number of sources.5,6 The most extensively studied, and discussed here, are those of Ps. stuzeri, P. halodenitrificans, and P. denitrificans. EPR and variable-temperature MCD studies of oxidized PsNOR showed the presence of three 6-c heme groups with distinct axial coordination.90,95
Two of these are magnetically isolated low-spin ferric heme c and heme b, which have IR transitions characteristic of bis-His and His-Met ligation. The third heme, b3, has unusual temperature-dependent spin state and EPR and MCD spectroscopic features consistent with
His/OH coordination. This differs from the penta coordination assigned to heme b3of oxidized PdNOR from room temperature resonance Raman data.95
The high-spin ferric heme b Fe and FeB are strongly antiferromagnetically coupled which results in both metal centers being EPR silent.88,90,94The nature of this interaction was defined in a resonance Raman study of oxidized PdNOR.96 When incubated with 18O-labeled water the
16O–18O difference spectrum showed a vibration at830 cm1reduced in intensity. Comparison with the model porphyrin compound97 tris(2-(5L)FeIII–O–FeIII–Cl)þ identified the as band as arising from a (FeOFe) grouping with a FeFe separation of3.5 A˚. No effect of D2O on the spectra was observed, suggesting that the oxo-bridge is not protonated. On reduction of the center the bridging oxygen leaves as water and the high-spin heme Fe coordinates to histidine, resulting in a site poised to bind NO.96
8.28.4.1.1 Structural model
Astructural model for NorB has been proposed, based on the crystal structure ofR. denitrificans CcO and the conserved primary structures of three NorB sequences from different organisms.3,6 Although the overall sequence homology is only 18%, the six His residues that bind the heme and CuB of CcO are conserved. Constraints for the relative positions of these His ligands of the high- and low-spin hemes and the CuA (FeB) centers were applied, which allowed a model based on multiple alignment of the sequences to be developed. The two hemes are nearly perpendicular to the membrane surface, and the nonheme FeB linked via an oxo-bridge to the high-spin Fe ion heme b3. Because the preferred coordination geometry of FeIII is octahedral or penta/hexa geometry the additional ligands are proposed to be conserved Gln residues.6 Mutagenesis of PdNOR has shown two of these residues to be important for activity with a suggested role, not in ligation, but in proton delivery or as a base involved in NO redox chemistry.98 Electron cryo- microscopy of two-dimensional crystals of PdNOR shows similarities with CcO indicating a common molecular architecture.89Aschematic of the structure of NOR is shown in Figure 9.
The topology of the redox centers is consistent with electron transfer rates described in Section 8.28.4.1.3. Since the rate of recombination of CO with NOR is some three orders of magnitude faster than with the active site of CcO, this suggests a more open environment of the heme in NOR. CO binds to the ferrous heme b3 to form a 6-c low-spin adduct.99 Laser flash photolysis results in cleavage of the FeCO bond, followed by recombination of the CO at a rate of konẳ1.7108M1s1. In contrast, the rate of formation of the initial CO adduct of NOR is much slower (konẳ1.2105M1s1) possibly due to the displacement of a distal heme ligand (OH/H2O) to allow binding of CO.
8.28.4.1.2 Mechanistic studies
Studies of ligand binding to NOR and the kinetics of these processes benefit from comparative studies on heme copper oxidases of known structure. Spectroscopic studies suggest that the spin- coupled dinuclear iron center, comprised of the hemeb3–FeBentity, is the active site of NOR, and the low-spin hemes band chave a role in electron transfer to the active site.
Fe3+ Fe3+
His
His His His Heme b3 FeB Fe3+
His
Heme b Fe3+ His
His
Heme c Met
O
low-spin Em = 345 mV
high-spin Em = 60 mV EPR silent low-spin
Em = 310 mV Em = 320 mV
EPR silent Nor B
Nor C
Figure 9 Schematic of the organization of the redox centers of prototype NOR.
The addition of NO to ascorbate-reduced NOR results in the formation of two strong EPR signals atgẳ2 and 4 due to the disruption of the magnetic coupling of the ferrous high-spin heme and the ferrous FeBatom. The complex signal atgẳ2 with a peak atgẳ2.08 is typical of nitrosyl ferroheme. The other signal, withgẳ4.11, 3.95, and 2.0, was assigned to a Sẳ3/2 spin system arising from a nonheme FeIINO adduct. Thus, under turnover conditions the two ferrous iron atoms at the active site both bind NO, behaving like mononuclear centers.100
The kinetics of NO binding to hemeb3has been determined by optical flow-flash methods, in which the reaction is initiated by the photolysis of CO bound to hemeb3.89In the first phase of the subsequent reaction with NO (after 2ms) a spectral species characteristic of ligand binding to a high-spin heme is formed. It has been suggested that NO partitions into the lipid phase generating higher localized [NO]
around the enzyme and that FeBprovides a second binding site, consistent with EPR data.
8.28.4.1.3 Heme–heme electron transfer
Equilibrium titration of PdNOR showed theEm value of FeB was 320 mV, that of hemeb3was þ60 mV, and those of hemecand hemebwereþ310 mV andþ340 mV, respectively.100The rates of intramolecular electron transfer between the redox centers of NOR have been investigated using the
‘‘electron backflow’’ technique.89The photolysis of CO bound to the high-spin hemeb3results in a decrease inEmof the heme to its basal value, causing a transient redistribution of electrons with the other redox centers before CO rebinding occurs. Time-resolved optical spectroscopy and electro- metric changes in membrane potential89 induced by photolysis of the CO-bound mixed-valence state of PdNOR reconstituted into proteoliposomes indicate that the pathway of electron transfer is similar to that of the heme Cu oxidases, consistent with the modeling studies discussed above.
Electrons are accepted from external donors by heme c, transferred to the low-spin heme d(kobsẳ3104s1), and then to hemeb3of the binuclear center (kobs>106s1). This technique revealed that FeBremained reduced and that electron backflow involved only the heme centers.
The separation of the redox potentials has enabled the three-electron reduced state with FeIII heme b3 of PdNOR to be characterized spectroscopically.100 It was proposed that the thermo- dynamic barrier presented by the lower potential of hemeb3prevents the two-electron reduction of the binuclear center to avoid the formation of a stable inactive FeII–heme b3 NO adduct.
However, a much smaller difference between Em of hemecand heme b3was deduced from the equilibrium constant for electron transfer between the two hemes in the electron backflow experiments101 indicating that, as with CcO, the redox centers exhibit negative cooperativity.
Thus reduction of the redox center with the lowestEmis more difficult when the other centers are already reduced resulting in an apparent decrease ofEmof the center.
8.28.4.1.4 Substrate reduction
Two modes of binding of NO have been considered, one involving binding of two NO molecules to a single metal site (the ‘‘cis’’ mechanism) and the other involving binding of NO to both metals of a binuclear site (the ‘‘trans’’ mechanism). The former mode clearly applies in the case of catalysis by FoP450nor, where only a single heme site is present (seeSection 8.28.5) and it has also been proposed for bacterial NORs. Spectroscopic studies of the reaction of NO with cytochrome bo3 were consistent with the formation of a CuBII(NO)2 species.102 The role of heme o3 in the subsequent formation of N2O was proposed to be that of abstraction of an O atom to form an oxyferryl species, a mechanism that avoids the formation of a stable FeIINO species. The close relationship of cytochrome bo3 with NOR led to the proposal that these enzymes share a common mechanism for N2O formation,103 and due to the relatively low Em
of hemeb3relative to hemes cand bformation of a stable FeIINO would be minimized.
EPR changes on binding NO to reduced PdNOR were consistent with both ferrous iron atoms of the binuclear heme b3 FeB binding NO.89 A‘‘trans’’ catalytic cycle for PdNOR has been proposed in which hemeb of the reduced catalytic center has His ligation. On binding two NO molecules cleavage of the Fe—His bond occurs.95On elimination of N2O from the FeII–NO::NO–
FeIIspecies the two Fe atoms are oxidized and become bridged by an oxo group. On reduction by internal electron transfer the bridging ligand is lost and is replaced by the original His ligand96 (seeFigure 10). This scheme apparently conflicts with the report that the rapid initial binding of NO to hemeb3(within 2ms, studied by flash-flow) does not significantly perturb the proximal His
heme ligand.101However, it is possible that a transient 6-c species with His/NO ligation is formed as has been reported for the interaction of NO with guanylate cyclase104and cytochromec0.105
Electrometric and spectrophotometric time-resolved studies of proton and electron flow in PdNOR used ‘‘flow-flash’’ laser activation of the CO–heme b3, fully reduced enzyme to initiate turnover both in proteoliposomes and of soluble preparations.101Subsequent changes in potential generated by the movement of charge across the membrane dielectric in proteoliposomes were complex, and were fitted to a sequential reaction model with six phases. Under these conditions fully reduced NOR has sufficient electrons for two turnover events, and the primary event is most likely to involve the reduced active site. The first rapid change was the formation of a ferrous hemeb3–NO adduct (kobsẳ5105s1) and a suggested binding of NO to FeB. This observation is not consistent with a ‘‘cis’’ model analogous to that involving CuAproposed for CcO.103
8.28.4.1.5 Model studies
Agood structural and functional model of the heme/nonheme di-iron center of the active site of the prototype NOR has been developed and characterized by Karlin and co-workers.14,97,106The complexes consist of a binucleating ligand 5L with a tetradentate tris(2-pyridylmethyl)amine moiety tethered to a synthetic tetraarylporphyrin (with three 2,6-difluorophenyl meso substitu- ents; seeFigure 11). As discussed above, the resonance Raman spectra of the oxidized compound show features in common with NOR supporting the assignment of the active site as oxo-bridged in the oxidized enzyme.
Reduction of the [(5L) FeIIIOFeIII—Cl]þ complex results in the loss of the oxo-bridge to form [(5L) FeIIFeII–Cl]þ, which shows reactivity towards NO. At low [NO], N2O is formed and the original oxo-bridged complex reformed, providing a good functional model for NOR function (Figure 11). At high [NO] the reaction is more complex producing a stable ferrous nitrosyl adduct and NO2in addition to N2O. Details regarding the site(s) at which NO binds to the complex are not available, since no stable intermediates could be trapped at80C.