Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity

A vital role has been identified for the heme-lysine cross-link unique to cytochromes P460: preventing enzyme deactivation during catalysis by the obligate nitrification metabolite nitric oxide.


Introduction
Ammonia (NH 3 )-oxidizing bacteria (AOB) derive energy for life from nitrication: the proton-coupled multi-electron oxidation of NH 3 to nitrite (NO 2 À ). 1,2 Nitrication begins with the oxidation of NH 3 to hydroxylamine (NH 2 OH) by the integral membrane enzyme ammonia monooxygenase. NH 2 OH is then oxidized to nitric oxide (NO) by the multi-heme enzyme hydroxylamine oxidoreductase (HAO) to establish net electron ow. 3 The physiological means through which NO is oxidized to NO 2 À are unknown. Detailed mechanistic understanding of controlled NH 2 OH oxidation is vital to the understanding of how nature uses NH 3 as fuel.
HAO is a homotrimer of octaheme subunits. 4 Seven of the hemes in each subunit are coordinatively saturated c-type hemes that mediate electron transfer. The remaining heme, also a c-type heme, is the site of NH 2 OH oxidation. This heme is called the heme P460 center because it has a characteristic Fe II Soret absorption maximum at 463 nm. 5 HAO heme P460 cofactors are unique in that they feature two cross-links with a tyrosine (Tyr) side chain ( Fig. 1a and b). The Tyr C3 1 and phenolate O cross-link with the c-heme at the 5 0 meso carbon and the adjacent pyrrole a-carbon, respectively. 4 These attachments disrupt the p conjugation of the porphyrin ring and distort the planarity of the heme, resulting in a ruffled heme structure. P460 cofactors are found within enzymes from other bacteria as well, including methanotrophs 6 and anaerobic NH 3 oxidizers (anammox). 7 The anammox bacterium Kuenenia stuttgartiensis contains at least 10 P460-containing HAO paralogs, at least two of which exhibit hydroxylamine or hydrazine oxidoreductase activity. 7,8 Thus, the presence of P460 cofactors appears to be a hallmark of N-oxidation functionality. 9 However, the characteristics that make this cofactor suitable for such reactions remain unknown.
Detailed understanding of NH 2 OH oxidation by heme P460 centers is necessary to establish a link between the properties of this unique cofactor and its reactivity. However, spectroscopic probing of reaction intermediates at the 3 HAO heme P460 cofactors is occluded by the signals of the 21 electron transfer hemes. To overcome this challenge, we have explored the NH 2 OH oxidation mechanism of cytochrome (cyt) P460, a soluble, dimeric mono-heme enzyme found in the periplasms of AOB. 9 In contrast to HAO, cyt P460 cofactors feature a Lys side chain amine covalently attached to the 13 0 meso C of the c-heme ( Fig. 1c and d). 10 Despite this substitution, the UV-visible (UVvis) absorption and 57 Fe Mössbauer properties characteristic of Fe II heme P460 are preserved. 11 Cyt P460 had previously been reported to oxidize NH 2 OH to NO 2 À . 12 However, we recently showed that cyt P460 oxidizes NH 2 OH selectively to nitrous oxide (N 2 O), not NO 2 À , under anaerobic conditions. 13 In our reported working mechanism (Fig. 2), Fe III cyt P460 binds NH 2 OH to form a stable Fe III -NH 2 OH adduct. This species then undergoes 3-electron oxidation to an {FeNO} 6 intermediate (Enemark-Feltham 14 notation denoting either Fe III -NOc, Fe II -NO + , or Fe IV -NO À ). Nucleophilic attack on this {FeNO} 6 intermediate by a second equivalent of NH 2 OH produces N 2 O. Consequently, each NH 2 OH is oxidized by 2 electrons. Accounting for 2-electrons recycled to NH 3 monooxygenase for turnover, 1 such a cycle does not generate net cellular reducing equivalents for AOB. The aforementioned reactivity prompted our reevaluation of the bacterial nitrication pathway. We noted that NO 2 À was among the products of cyt P460 reactivity with NH 2 OH when the reactions were carried out aerobically. 13 However, NO 2 À concentrations were never stoichiometric with NH 2 OH input, likely owing to competition with the reaction producing N 2 O. We hypothesized that this NO 2 À was produced as a result of the non-enzymatic reaction of aqueous NO with O 2 , 15 implying that P460-driven NH 2 OH oxidation terminates at the {FeNO} 6 intermediate. If NO loss could outcompete the reaction of this intermediate with NH 2 OH, the 3-electron oxidation of NH 2 OH would be assured and, thus, establish a net electron ow for AOB. Subsequent experiments conrmed that this outcome indeed occurs with HAO: NH 2 OH is enzymatically oxidized by 3 electrons to NO and then swily released. 3 Thus, rapid access to the {FeNO} 6 intermediate is essential for the N 2 O-generating mechanism of cyt P460 and the NOgenerating mechanism of both cyt P460 and HAO. However, concerted biological 3-electron oxidation is unlikely. Moreover, the cyt P460 {FeNO} 6 species is formed with either 1-or 2-electron oxidants. 13 These observations strongly suggest that the oxidation of Fe III -NH 2 OH to the {FeNO} 6 intermediate occurs via sequential 1-or 2-electron steps, or both. Proposed intermediates include a 1-electron-oxidized Fe III -NH 2 OH radical (Fe III -cNH 2 OH) and a 2-electron-oxidized species formulated either as a ferric nitroxyl (Fe III -HNO) or its conjugate base {FeNO} 7 . 16 However, no evidence for these intermediates has been provided. Electron paramagnetic resonance (EPR) spectroscopy provided evidence for a minor 5-coordinate (5c) {FeNO} 7 species formed either when preparing Fe III -NH 2 OH samples or following complete oxidant consumption aer multiple turnovers of cyt P460. 13 This species was shown to be off-pathway. Hendrich and co-workers 17 observed a similar offpathway 5c {FeNO} 7 species with EPR spectroscopy when fully reduced HAO was allowed to react with NH 2 OH.
Herein, we report the characterization of a 6-coordinate (6c) {FeNO} 7 intermediate in N. europaea cyt P460 that is on-pathway and precedes the formation of the critical {FeNO} 6 species. This 6c {FeNO} 7 intermediate slowly decays in a NO-independent manner to the off-pathway 5c {FeNO} 7 species. This conversion represents dissociation of the axial His140. Kinetic studies of a 13 0 cross-link-decient cyt P460 mutant (Lys70Tyr cyt P460) revealed that at least one function of this cross-link is to kinetically bypass the production of the off-pathway 5c {FeNO} 7 intermediate during turnover by protecting the cofactor from deactivation by NO. The rate of 6c-to-5c conversion in the Lys70Tyr cyt P460 {FeNO} 7 is accelerated by several orders of magnitude compared with the wild-type (WT) protein due to the mechanistic participation of excess NO. This rapid, NOdependent 6c-to-5c {FeNO} 7 conversion is reminiscent of the activation mechanism for heme-NO/O 2 (H-NOX) binding proteins including soluble guanylate cyclase (sGC). 18 Fig. 1 Views of the top (a) and side (b) of the Nitrosomonas europaea HAO heme P460 cofactor (2.1Å resolution X-ray crystal structure, PDBID 4FAS) and the top (c) and side (d) of the N. europaea cytochrome (cyt) P460 heme P460 cofactor (1.8Å resolution X-ray crystal structure, PDBID 2JE3). Both cofactors are c-heme cofactors with additional covalent amino acid side chain attachments. In HAO, Tyr467 from a neighboring subunit cross-links via the C3 at the 5 0 meso carbon of the porphyrin and via the phenolate O at the neighboring pyrrole a-carbon. In cyt P460, the Lys70 amine N cross-links to the 13 0 meso carbon.

species
In our previous study, 13 we generated an off-pathway 5c {FeNO} 7 species. This species was generated via the treatment of Fe III cyt P460 with the HNO donor disodium diazen-1-ium-1,2,2 triolate (Na 2 N 2 O 3 ). One mole of Na 2 N 2 O 3 liberates 1 mol of HNO with a half-life of 2 min when in room temperature pH 8.0 buffer. Hereaer, all HNO concentrations are expressed as the nominal nal concentration expected from this Na 2 N 2 O 3 decomposition. In the present work, monitoring of the UV-vis absorption spectral time course immediately aer the treatment of 15 mM Fe III cyt P460 with 100 mM HNO revealed a previously uncharacterized species. The new species forms within 2 min and exhibits a UV-vis absorption spectrum with a Soret maximum at 452 nm and Q-band maxima at 550, 608, and 665 nm (Fig. 3). This species decays within 15 min, resulting in a UV-vis absorption spectrum with a Soret maximum at 455 nm and Qband maxima at 535, 584, and 642 nm. These absorption features correspond to the aforementioned 5c {FeNO} 7 species. 13 A similar spectral time course was observed when 15 mM Fe II cyt P460 was treated with 100 mM NO generated by Proli-NONOate at pH 8.0. One mole of Proli-NONOate liberates 2 mol of NO with a half-life of 2 s in room temperature pH 8.0 buffer. Hereaer, all NO concentrations are expressed as the nominal nal concentration expected from this Proli-NONOate decomposition. Isosbestic points observed in these spectral time courses at 430, 556, 600, 610, and 652 nm indicate a onestep conversion between the two species. Together, the data suggest that an uncharacterized species forms and slowly decays to the 5c {FeNO} 7 species.
The spin state of this new species was characterized with continuous-wave X-band EPR (Fig. 4). Samples were prepared by treating 150 mM cyt P460 with 750 mM HNO in pH 8.0 buffer at 25 C. The samples were frozen with liquid N 2 within 3 min of mixing. The resulting EPR spectrum was consistent with an S ¼ 1/2, 6c heme {FeNO} 7 species: the simulated g-values were 2.10, 2.01, and 1.98 with corresponding 14 N hyperne values of 37, 55, and 40 MHz, respectively. 19 The sample under the same reaction condition but frozen aer 1 h had a distinct EPR spectrum with simulated g-values of 2.10, 2.03, and 2.01 and corresponding 14 N hyperne values of 50, 57, and 45 MHz, respectively. These parameters match those previously reported for the off-pathway 5c {FeNO} 7 intermediate. 13 Typically, 6c {FeNO} 7 complexes exhibit a 9-line 14 N superhyperne splitting from the bound NO and the axially bound N(His). The lack of a 9-line 14 N superhyperne splitting from the bound Fe-N(His) could indicate either a weak Fe-N(His) bond or a large degree of . In (a), the solid red trace is the spectrum collected immediately after mixing, the solid black trace is the spectrum collected after 20 min, and grey spectra were collected in 30 s increments. The inset highlights the time course in the Q-band region. Absorption maxima in nanometers are labeled with colors corresponding to each species. Isosbestic points are labeled in gray. In (b), the black trace is a single exponential (A 452 ¼ y 0 + A Â e ÀkobsÂt ) fit to the data, yielding k obs ¼ 3.15 Â 10 À3 s À1 .  disorder of the bound His. 20 However, complementary spectroscopic data using techniques such as electron nuclear double resonance (ENDOR) would need to be acquired in order to accurately quantify the Fe-N(His) hyperne interaction. These investigations are underway and will be reported elsewhere.
Fe K-edge X-ray absorption spectroscopy (XAS) data were obtained for both of these {FeNO} 7 species. The Fe-K edge absorption near-edge regions of both the 6c and 5c {FeNO} 7 species are shown in Fig. 5. The pre-edge feature near 7113 eV is conventionally assigned as a quadrupole-allowed Fe 1s / 3d transition that can gain intensity via an electric dipole mechanism. 21 This feature appears at 7113.3 eV in the spectrum of the initially formed 6c {FeNO} 7 intermediate. The feature exhibits signicantly higher intensity in the 5c {FeNO} 7 spectrum, and this intensity increase is consistent with decreased centrosymmetry at Fe: as the coordination number decreases, the attendant diminished centrosymmetry confers dipole allowedness to the 1s / 3d transition and a corresponding increase in pre-edge intensity.
Fits of the extended X-ray absorption ne structure (EXAFS) region from k values of 2-14Å À1 , where k is the photoelectron wave number, for the WT 6c and 5c {FeNO} 7 species give short Fe-N scatters assignable to Fe-NO at 1.86Å and 1.74Å, respectively (Fig. S9 † and Table 1). These distances are consistent with typical Fe-NO bond lengths in 6c and 5c heme {FeNO} 7 species, respectively. 22 The data resolution precluded the tting of an Fe-N(His) scattering path in the 6c {FeNO} 7 species independent from the Fe-N(heme) paths. However, the EXAFS were best t for this species with 5 rather than 4 Fe-N scatters at 2.04Å. Moreover, we could reliably t the axial His of the 5c {FeNO} 7 intermediate at a distance 2.53Å, which is well outside the range of coordination.
The combined UV-vis absorption, EPR, and X-ray absorption data reveal that Fe III cyt P460 reacts with HNO to accumulate a 6c {FeNO} 7 species, which subsequently decays to a 5c {FeNO} 7 form within 30 min. The simplest interpretation of this data is that the conversion results from the dissociation of the axial His140. Dissociation of the ligand trans to the NO is frequently observed for heme {FeNO} 7 species and is attributed to the trans inuence of NO. [23][24][25][26][27] Cyt P460 6c {FeNO} 7 7 . The addition of 3 mM NH 2 OH to this solution resulted in no changes to the UV-vis absorption spectrum or the rate of 6c-to-5c {FeNO} 7 conversion (Fig. S1 †). These results suggest that NH 2 OH is unreactive with the 6c {FeNO} 7 species.
By contrast, oxidant addition promoted rapid changes in the UV-vis absorption spectrum. Anaerobic treatment of 15 mM cyt P460 6c {FeNO} 7 with 100 mM of PMS [phenazinemethosulfate, 7 decay within 30 s (Fig. 6). The product of this reaction had UV-vis absorption features identical to those assigned to the cyt P460 {FeNO} 6 species. 13 Previous experiments showed that treating cyt P460 5c {FeNO} 7 with these oxidants afforded no evidence of {FeNO} 6 formation. 13 The only spectral changes observed were a minor decrease in the 455 nm Soret maximum and the appearance of a shoulder at 414 nm, which suggested cofactor degradation ( Fig. S2 †). No conversion to {FeNO} 6 was observed even aer treatment with the far more potent oxidant potassium hexachloroiridate K 2 [IrCl 6 ] (E 0 ¼ +892 mV vs. NHE). Here again, only degradation occurred.
EPR spectra obtained for cyt P460 6c and 5c {FeNO} 7 samples treated with oxidant ( Fig. S3a and b †) corroborated the results of the UV-vis absorption experiments. Under anaerobic conditions, 600 mM PMS was added to a solution of 200 mM 6c cyt P460 {FeNO} 7 at pH 8.0 and 25 C, and the mixture was immediately frozen in liquid N 2 . The resulting EPR spectrum was dominated by a sharp signal at g ¼ 2.0 attributed to PMS semiquinone. The spectrum lacks features assigned to the 6c {FeNO} 7 species and shows no evidence for any other Fe-based signals. This outcome suggests that most of the cyt P460 Fe is in an EPR-silent state, which is consistent with the oxidation of the 6c {FeNO} 7 species to the EPR-silent {FeNO} 6 form. Moreover, the EPR spectrum obtained aer the addition of 600 mM PMS to 200 mM 5c cyt P460 {FeNO} 7 at pH 8.0 and 25 C shows EPR features consistent with those of the 5c {FeNO} 7 species as well as the g ¼ 2.0 signal assigned to PMS semiquinone. This PMS semiquinone spectrum is observed in samples of PMS in buffer, indicating that the semiquinone form is present even in the absence of protein or NH 2 OH. The aggregate data are consistent with the 5c {FeNO} 7 species being unreactive and off-pathway; however, the 6c {FeNO} 7 species can be oxidized to {FeNO} 6 . The 5c species therefore must have a more positive reduction potential than the 6c {FeNO} 7 . We rationalize that this is largely attributable to electron donation from His producing a more electron-rich FeNO unit. These data suggest that 6c {FeNO} 7 is an intermediate of NH 2 OH oxidation by cyt P460.
To demonstrate conclusively that cyt P460 6c {FeNO} 7 is a NH 2 OH oxidation intermediate, we generated this species by oxidizing the Fe III -NH 2 OH species. The 6c-to-5c {FeNO} 7 conversion occurs on a minutes timescale, whereas oxidant rapidly converts this species to {FeNO} 6 on a seconds timescale. Therefore, if 6c {FeNO} 7 is generated as a catalytic intermediate, the presence of oxidant will kinetically favor its conversion to {FeNO} 6 over His dissociation to form the off-pathway 5c {FeNO} 7 species. Oxidation to {FeNO} 6 should be rst-order with respect to oxidant concentration. Therefore, at low oxidant concentrations, the rate of the oxidation pathway will be slow enough for the His dissociation pathway to kinetically compete. By these rationales, the 6c {FeNO} 7 intermediate should accumulate immediately aer the depletion of the oxidant. To test this hypothesis, we allowed 200 mM cyt P460 to react with 1 mM DCPIP and 1 mM NH 2 OH at pH 8.0 and room temperature. The sample was frozen in liquid N 2 within 2 min, a time immediately aer the blue color of the DCPIP disappeared. The 20 K EPR spectrum of this sample exhibited one anisotropic S ¼ 1/2 signal with features identical to those observed for 6c {FeNO} 7 (Fig. S4 †). The signal accounts for 40 mM or 20% of the Fe centers in the sample. In the absence of any other Fe-based signal, we accounted for the remainder of the iron (160 mM) as EPR-silent {FeNO} 6 . Our inability to detect the 6c {FeNO} 7 species within UV-vis absorption time courses likely results from the low ratio of 6c {FeNO} 7 to {FeNO} 6 concentrations and the overlapping UV-vis absorption features of the two species. Nevertheless, the EPR spectra clearly show evidence for the formation of the 6c {FeNO} 7 species, and therefore, we assigned 6c {FeNO} 7 as an intermediate on the cyt P460 NH 2 OH oxidation pathway.

NO-independent His140 dissociation from cyt P460 {FeNO} 7
NO promotes rapid (milliseconds to seconds) His dissociation in many heme proteins. 18,20 This phenomenon underlies the mechanism of signal transduction by H-NOX proteins and sGC in both eukaryotes and bacteria. 28 Given this common behavior by heme proteins, we sought to test if the rate of His dissociation from the cyt P460 6c {FeNO} 7 is also promoted by NO.
The cyt P460 6c {FeNO} 7 species can be generated from the reaction of either Fe III cyt P460 with HNO or Fe II cyt P460 with NO ( Fig. S5 † and 7). The two independent methods allows for testing the reactivity of 6c {FeNO} 7 in the absence or presence of a large excess of NO, respectively. Rate constants for 6c-to-5c {FeNO} 7 conversion were obtained from the reaction of 15 mM Fe II cyt P460 with varying excess concentrations of NO ranging from 100-600 mM. The conversion was monitored by the  (Fig. 7). The data clearly show that His140 dissociation for cyt P460 6c {FeNO} 7 proceeds via a mechanism that is independent of either NO or HNO. This behavior contrasts starkly with the behavior common to NOsensing heme proteins. Furthermore, the His dissociation of cyt P460 is appreciably slower than that observed for other heme proteins, which dissociate their axial His on millisecond time scales. This slow His dissociation allows the oxidation of 6c {FeNO} 7 to {FeNO} 6 to kinetically outcompete the formation of the off-pathway 5c {FeNO} 7 intermediate, and thus, appears essential to preserving active catalyst.
Characterization of {FeNO} 7 species on a cross-link decient mutant, Lys70Tyr cyt P460 The lack of an NO-dependent k His-off for cyt P460 6c {FeNO} 7 prompted us to investigate how this anomalous behavior relates to the unique P460 cofactor structure. To this end, we hypothesized that the distinguishing Lys70 cross-link to the 13 0 meso C of the P460 cofactor may inuence k His-off . Therefore, we generated a cross-link-decient Lys70Tyr cyt P460 mutant for a comparison of His140 dissociation kinetics. Puried Fe III Lys70Tyr cyt P460 is a red protein with a UV-vis absorption Soret maximum at 406 nm and Q-bands at 500 nm and 632 nm (Fig. 8a). The continuous-wave X-band EPR spectra of the resting Fe III exhibited an S ¼ 5/2 signal with g-values of 5.78 and 1.98 (Fig. 8b). These g-values are consistent with an axial (E/D ¼ 0.00) signal and suggest an increased heme symmetry compared with the WT Fe III cyt P460 S ¼ 5/2 spectrum with an E/ D of 0.03. This increased symmetry is consistent with the loss of the Lys cross-link in the mutant.
To characterize the cofactor in the Lys70Tyr variant further, we obtained resonance Raman (rR) spectra via excitation near the Soret maxima of Fe III WT (l ex ¼ 457.8 nm) and Fe III Lys70Tyr (l ex ¼ 405.0 nm) cyt P460 (Fig. 8c). Detailed analysis of the Fe III WT cyt P460 rR spectrum was beyond the scope of the present work; however, the increased number of observed bands relative to non-cross-linked hemes suggests that the cyt P460 cofactor has diminished symmetry. This increase in band count is consistent with the rR spectrum obtained for the HAO Fe II heme P460 cofactor. 29 The rR spectrum obtained for Fe III Lys70Tyr cyt P460 exhibited an oxidation state marker band (n 4 ) at 1370 cm À1 but a spin-state marker band (n 3 ) at 1501 cm À1 . Typically, n 3 greater than 1500 cm À1 indicates a low spin 6coordinate heme, however, 5-coordinate ferric cyt c 0 proteinswhose coordination was veried by EPR spectroscopy and crystal structures-also exhibit n 3 ca. 1500 cm À1 . 30 The EPR spectrum of the Fe III Lys70Tyr cyt P460 is also consistent with a high-spin ferric heme. To further characterize the coordination number of the mutant cyt P460, we also obtained the rR spectrum obtained of Fe II Lys70Tyr cyt P460 (l ex ¼ 405.0 nm), which has the prole of a standard, effectively D 4h 5c high spin Fe II c-heme with n 4 at 1356 cm À1 and n 3 at 1473 cm À1 (Fig. S13 †). [30][31][32] The aggregate spectroscopic data are consistent with the restoration of a canonical c-heme due to loss of the 13 0 cross-link. 33 The Fe III Lys70Tyr cyt P460 binds NH 2 OH to form the Fe III -NH 2 OH adduct (Fig. S14 †). However, this mutant protein is Fig. 7 Plot of k obs vs. NO concentration (red circles) or HNO concentration (blue triangles). The corresponding k His-off values are 2.9 AE 0.2 Â 10 À3 s À1 and 5.7 AE 0.2 Â 10 À3 s À1 for HNO and NO, respectively. incapable of turnover, resulting from loss of the Fe III -NH 2 OH oxidation reactivity. Therefore, the cross-link is necessary for reactivity of the Fe III -NH 2 OH adduct. This lack of NH 2 OH oxidation reactivity in the mutant will be addressed elsewhere.
UV-vis absorption time courses of 6c {FeNO} 7 formation and decay were obtained to compare the His dissociation between WT and Lys70Tyr cyt P460. Anaerobic treatment of 10 mM Fe III Lys70Tyr cyt P460 with 600 mM HNO resulted in the appearance of a new species with an UV-vis absorption Soret maximum at 415 nm and Q-band maxima at 540 and 580 nm (Fig. 9). This species decayed slowly (within 80 min) to a species exhibiting a Soret peak at 413 nm with a shoulder at 396 nm and unshied Q-bands at 540 and 580 nm. By contrast, no intermediate was observed when 10 mM Fe II Lys70Tyr cyt P460 was treated with 600 mM NO. The UV-vis spectral time course showed the immediate formation of a stable species within the time of manual mixing. The absorption spectrum of this product matches that of the HNO reaction product, which suggests an identical Fe product for both reactions.
EPR spectroscopic analyses afforded informative characterizations of the two observed species. An anaerobic sample was prepared containing 150 mM Fe III Lys70Tyr cyt P460 and 700 mM HNO at pH 8.0 and 25 C. The sample was incubated for 3 min and frozen in liquid N 2 . The EPR spectrum of this sample exhibited g-values of 2.09, 2.02, and 1.98 with corresponding 14 (Fig. 10). As with the WT experiments, the differences in the two EPR spectra are consistent with a conversion from a 6c to a 5c {FeNO} 7 . The Lys70Tyr also does not exhibit a 9-line super hyperne splitting in the 6c {FeNO} 7 . The correlated EPR and UV-vis absorption spectra indicate that the reaction of Lys70Tyr Fe III cyt P460 with HNO forms a 6c {FeNO} 7 , which decays slowly to a 5c {FeNO} 7 .
Data for the Fe K-edge absorption near-edge regions and EXAFS region of the mutant 5c {FeNO} 7 species were collected for comparison with that of the WT. The Lys70Tyr mutant also exhibited a pre-edge feature near 7113 eV with intensity similar to that seen in the WT 5c {FeNO} 7 spectrum. This result is consistent with decreased centrosymmetry at the Fe center resulting from the decrease in coordination number from the dissociation of the axial His (Fig. S6 †). EXAFS data were collected to determine the bond distances of Lys70Tyr cyt P460 5c {FeNO} 7 , and ts of the EXAFS region from a k of 2-14Å À1 yielded a Fe-NO bond length of 1.80Å. As with that of the WT 5c {FeNO} 7 , the EXAFS data t best with the addition of the axial His as a separate parameter, yielding a bond length of 2.48Å, which is outside the range of coordination for an Fe-N(His) bond (Fig. S10 † and Table 1). Fig. 9 The 150 min UV-vis absorption full-spectral (a and b) and 415 nm single-wavelength time courses (c) of the reaction of 10 mM Fe III cyt P460 with 100 mM of HNO in 200 mM HEPES buffer (pH 8.0). In (a) and (b), the solid red trace is the spectrum collected immediately after mixing, the solid blue trace is collected at 10 min and the solid black trace was collected at 150 min. Grey spectra were collected in 1 min increments. The insets highlight the time courses in the Q-band region. Absorption maxima in nanometers are labeled with colors corresponding to each species. Isosbestic points are labeled in gray. In (c), the black trace is a double-exponential fit (A 415 ¼ A 0 + A 1 Â e Àkobs(1)Ât + A 2 Â e Àkobs(2)Ât ) to the data, yielding k obs(1) ¼ 3.7 Â 10 À3 s À1 and k obs(2) ¼ 2.7 Â 10 À4 s À1 .

Rapid, NO dependent His140 dissociation when cross-link removed
A 6c {FeNO} 7 species was observed when either Fe III WT or Lys70Tyr cyt P460 was allowed to react with HNO. However, this species was not observed during the reaction of the Fe II form of the mutant with NO, suggesting either the 6c {FeNO} 7 is never formed or it decays too fast for observation. To differentiate between these possibilities, we monitored the reaction of Fe II Lys70Tyr cyt P460 with NO using stopped-ow UV-vis absorption spectroscopy. The spectral time course exhibited accumulation within 20 ms of absorption features attributed to Lys70Tyr cyt P460 6c {FeNO} 7 (Fig. 11). This spectrum decayed within 5 s to a new spectrum characteristic of the 5c {FeNO} 7 species. An isosbestic point at 400 nm suggests direct conversion from the 6c to the 5c {FeNO} 7 species. These results verify that the 6c {FeNO} 7 species is generated when Fe II Lys70Tyr cyt P460 reacts with NO and its conversion to the 5c {FeNO} 7 species is rapid. This 6c-to-5c {FeNO} 7 conversion is orders of magnitude faster in the reaction when excess NO is present, suggesting that NO induces rapid His140 dissociation. Such rapid NO-dependent His dissociation from 6c heme {FeNO} 7 species has been previously characterized in several NO-sensing heme proteins.
Rate constants were determined for the NO-independent and NO-dependent His140 dissociation pathways of Lys70Tyr cyt P460 6c {FeNO} 7 . The NO-independent rate constant, k His-off , was determined from the reactions of 10 mM Fe III Lys70Tyr cyt P460 with HNO at various concentrations in the range of 100-600 mM. The accumulation and decay of 6c {FeNO} 7 was monitored at 415 nm using UV-vis absorption spectroscopy. Double-exponential functions were t to the 415 nm traces. The k obs for His140 dissociation was zeroth-order with respect to HNO (Fig. 12a). An averaging of the k obs values at all HNO concentrations provided a k His-off of 3.8 AE 0.9 Â 10 À4 s À1 , which is an order of magnitude slower than the k His-off measured for WT cyt P460.
The rate constant of the NO-dependent His140 dissociation pathway was measured using stopped-ow UV-vis absorption spectroscopy. The NO-dependent rate constant for His140 dissociation, k His-off(NO) , was determined from the reactions of 10 mM Fe II Lys70Tyr cyt P460 with 100-800 mM NO. These stopped-ow kinetics experiments were monitored at 415 nm. Because the 6c {FeNO} 7 species formed completely within the stopped-ow mixing dead time, the kinetic traces were monophasic that were well t by single exponentials. Extracted values of k obs were t to a linear regression with eqn (1) (Fig. 12b): The best-t parameters were a k His-off(NO) of 790 AE 80 M À1 s À1 with a y-intercept, or k app , of 0.36 AE 0.04 s À1 . The rst-order dependence of 6c-to-5c {FeNO} 7 conversion has been used to support a mechanism involving a hypothetical trans-dinitrosyl {Fe(NO) 2 } 8 intermediate in other heme systems. 34 Our spectral time course is inconsistent with formation of an intermediate during this conversion. Furthermore, it is unclear from our data what k app represents. In our hands, the 6c-to-5c {FeNO} 7 conversion is irreversible, thus the non-zero value for k app likely reports the rate constant of an alternative pathway. One possibility is that this y-intercept represents the parallel NOindependent His dissociation pathway, k His-off . However, this value was independently measured to be vastly slower: 3.7 AE 0.4 Â 10 À4 s À1 . Another possibility is that the range of NO concentrations surveyed was insufficient to observe saturating behavior; such behavior has been noted in other studies of 6cto-5c heme {FeNO} 7 conversion. 35 Our data clearly show that removal of the cross-link introduces an NO-dependent His140 dissociation pathway. However, more detailed mechanistic work will be required to correctly interpret the observed nonzero y-intercept of the NO-dependent His dissociation pathway.
The kinetics thus revealed two distinct pathways for His140 dissociation from the Lys70Tyr cyt P460 6c {FeNO} 7 . One pathway is independent of NO with a rst-order rate constant similar to that observed for the WT cyt P460 6c {FeNO} 7 species while the second pathway is absent in the WT cyt P460. The results of these experiments suggest that in the presence of excess NO, the Lys70 cross-link of the P460 cofactor is necessary to inhibit the NO-dependent pathway, thereby allowing the oxidation of 6c {FeNO} 7 to {FeNO} 6 . These results identify at Fig. 11 The 32 s stopped-flow UV-vis absorption full-spectral (a) and 415 nm single-wavelength (b) time courses of the reaction of 10 mM Fe II cyt P460 with 100 mM NO in 200 mM HEPES buffer (pH 8.0). In (a), the solid red trace is the spectrum collected immediately after mixing. The black trace is the final spectrum collected at 32 s. Grey spectra were collected in 0.5 s increments. Absorption maxima in nanometers are labeled with colors corresponding to each species. An isosbestic point is labeled in gray. In (c), the black trace is a single exponential (A 415 ¼ y 0 + A Â e ÀkobsÂt ) fit to the data, yielding k obs ¼ 0.40 s À1 . least one function of the cross-link characteristic of P460 cofactors.
Activation analysis of His140 dissociation from WT and Lys70Tyr: insight into the role of the cross-link in 6c-to-5c {FeNO} 7

conversion
Determination of activation enthalpies (DH ‡ ) and entropies (DS ‡ ) for the NO-independent pathways provided insight into how the Lys70 cross-link inuences the His140 dissociation rate in the NO-dependent pathway. The results of the Eyring analysis of NO-independent and NO-dependent pathways for WT and Lys70Tyr cyt P460 are shown in Table 2. For both the WT and Lys70Tyr, the NO-independent pathway is dominated by the dissociation of His140. The 1 kcal mol À1 difference in DG ‡ between the mutant and the WT proteins (DDG ‡ ) accords with the 10-fold smaller rate constant for the NO-independent His dissociation of Lys70Tyr cyt P460 compared with that of the WT. The increased barrier to dissociation can largely be attributed to the increased DH ‡ of the mutant dissociation reaction. The difference in DH ‡ of 11 kcal mol À1 between the Lys70Tyr and WT cyt P460 proteins implies an increased Fe-N(His) bond dissociation energy in the former. The relatively small DS ‡ of the WT (0.4 AE 0.3 cal mol À1 K À1 ) is surprising for a dissociation mechanism, which would assume an overall gain in DS ‡ and therefore a larger DS ‡ . This increase in DS ‡ of 27.3 cal mol À1 K À1 in the Lys70Tyr contributes to lowering the overall DG ‡ of the NO-independent pathway to account for a difference of 1 kcal mol À1 rather than the approximately 9 kcal mol À1 difference if the mutant and WT shared a similar DS ‡ . Therefore, this difference in DS ‡ suggests one functional contribution of the cross-link (vide infra).
The activation parameters were also obtained for the NOdependent His140 dissociation in Lys70Tyr cyt P460. Both the NO-dependent and NO-independent pathways for Lys70Tyr cyt P460 yield similar DH ‡ values. This is consistent with the rate of the 6c-to-5c {FeNO} 7 conversion for both pathways being dominated by the Fe-His bond dissociation energy. The entropic terms from the NO-dependent and NO-independent pathways differ by approximately 30 cal mol À1 K À1 , a value that commonly attends a unit change in reaction order. 36

Conclusions
We have identied a 6c {FeNO} 7 intermediate on the cyt P460 NH 2 OH oxidation pathway (Fig. 13). This species results from an apparent 2-electron oxidation of Fe III -NH 2 OH cyt P460. We suspect that Fe III -NH 2 OH conversion to 6c {FeNO} 7 occurs via two subsequent and rapid 1-electron oxidation steps with reduction potential inversion between these steps. 38 Possible 1electron oxidized intermediates are either the Fe III -cNH 2 OH, invoked as an intermediate for cyt P450 nitric oxide reductase Fig. 12 Plots of k obs for 6c-to-5c {FeNO} 7 vs. HNO concentration (a) and NO concentration (b). The corresponding rate constants are k His-off ¼ 3.8 AE 0.9 Â 10 À4 s À1 and k His-off(NO) ¼ 790 AE 80 Â M À1 s À1 for HNO and NO, respectively.  6 proceeds far more swily than His dissociation to form the 5c {FeNO} 7 species. Thus, in the presence of oxidant, catalysis outpaces the formation of the irreversible, off-pathway 5c {FeNO} 7 , thereby preserving active enzyme. From this result, we speculate that the in vivo lifetime of cyt P460 is partially dependent on steady-state oxidant concentrations, which should uctuate with periplasmic O 2 concentrations. This mechanism could affect active cyt P460 concentrations when AOB transition from oxic to anoxic environments. The bulk enzyme could tolerate short periods of low O 2 concentration given that 6c-to-5c {FeNO} 7 conversion requires several minutes. Determining if HAO inactivates in a similar fashion upon oxidant depletion could determine whether our hypothesis also applies to energy-producing reactions in AOB. Indeed, a 5c {FeNO} 7 species of the HAO P460 cofactor has been observed when the reduced enzyme is treated with NH 2 OH. 17 However, it remains unclear whether this species is similarly unreactive, as observed for cyt P460 5c {FeNO} 7 intermediate.
Experiments with the Lys70Tyr cyt P460 showed how the Lysheme cross-link, the dening characteristic of cyt P460s, is critical for avoiding the off-pathway 5c {FeNO} 7 . This cross-link lengthens the lifetime of the 6c {FeNO} 7 intermediate (k His-off ¼ 2.9 Â 10 À3 s À1 ) compared to that of the other heme proteins. As noted above, the lifetime of the 6c {FeNO} 7 is related to the trans inuence exerted by the NO, which weakens the Fe-His140 bond. However, we noted that the DH ‡ for His dissociation is larger for the mutant than the WT, implying the mutant has a stronger Fe-N(His) bond strength. The stronger Fe-N(His) bond strength in Lys70Tyr is consistent with the loss of heme ruffling aer the removal of the cross-link; the restoration of planarity allows electron delocalization from Fe into the porphyrin p system. 41 Consequently, Fe becomes less electronrich, strengthening the Fe-N(His) interaction while weakening the Fe-NO interaction. The elongated Fe-NO distance of the . In other words, one product of cyt P460 is NO. In the absence of an NO sink, cyt P460 could be surrounded by a local pocket of high NO concentration generated by its own turnover; thus, cyt P460 generates a potential poison to its own catalytic cycle. Furthermore, recent work from our laboratory has established NO as an obligate intermediate in the NH 3 -oxidizing pathway through NH 2 OH oxidation by HAO. 3 Thus, intracellular NO likely accumulates during oxic metabolism. The cross-link, therefore, appears to be necessary for avoiding cyt P460 catalysis inactivation under conditions with available NO.
NO-dependent 6c-to-5c {FeNO} 7 conversion is also characteristic of the human NO-sensing protein sGC. Along with its bacterial counterparts, these NO sensors are collectively known as H-NOX (Heme NO/O 2 binding) proteins because they contain an H-NOX domain, a b-heme with an axially bound His. Downstream signaling is conferred through a partner protein, or in sGC, an attached guanylate cyclase domain. NO binds to the Fe II -heme to form a 6c {FeNO} 7 species that rapidly converts to a 5c {FeNO} 7 species. The ensuing His dissociation induces a protein conformational change that activates the cyclase domain, thereby initiating the signaling cascade. Three hypotheses have been offered to explain the observed NO dependence on the rate of the 6c-to-5c {FeNO} 7 conversion: (1) NO binding at an allosteric site promotes His dissociation, (2) a second NO molecule replaces the axial His to form a transdinitrosyl intermediate, or (3) nucleophilic attack on the {FeNO} 7 intermediate by a second NO molecule results in N-N bond formation, which in turn results in His dissociation. 18 However, NO dependence on His dissociation has also been observed for our cyt P460 mutant, cyt c 0 -a and an engineered heme/non-heme nitric oxide reductase, none of which are related to H-NOX proteins. 42,43 These non-H-NOX proteins are unlikely to contain the same allosteric site, therefore, we can rule out hypothesis 1 as a general mechanism. Biological N-N bond formation is preceded by diferrous-dinitrosyl ([{FeNO} 7 ] 2 ), Fe III -cNH 2 OH, or {FeNO} 6 intermediates. 13,40,42,[44][45][46] Although we cannot rule out hypothesis 3, N-N bond formation via the nucleophilic attack of NO on an {FeNO} 7 species has not yet been observed in a biological system. In support of hypothesis 2, the crystal structure of a cyt c 0 -a protein from Alcaligenes xylosoxidans, which also exhibits NO-dependent 6c-to-5c {FeNO} 7 conversion, shows that NO is bound to the proximal side of the heme. 47 Furthermore, kinetic evidence exists for an intermediate species between the 6c and 5c {FeNO} 7 of a Nostoc sp. H-NOX protein. 48 This intermediate was proposed to be trans-dinitrosyl. We saw no evidence for an intermediate in our experiments, which suggests that the mechanism for Lys70Tyr cyt P460 differs from those proposed for H-NOX. Therefore, to elucidate this NO-dependent mechanism, we will need to perform further kinetic and intermediate trapping studies. For the purpose of the current study, the relevant result is that the removal of the cross-link causes the rate of His dissociation to be dominated by a NO-dependent pathway that is absent in the native cyt P460.
The lack of a NO-dependent pathway for WT cyt P460 could be due to decreased accessibility at the proximal side of the heme, precluding the binding of the second NO molecule. For the NO-independent His140 dissociation pathway, the WT cyt P460 has a DS ‡ term (0.4 AE 0.3 cal mol À1 K À1 ) that is smaller than that for Lys70Tyr cyt P460 (27.6 AE 2.2 cal mol À1 K À1 ). A similarly large DS ‡ was found for His dissociation from A. xylosoxidans c 0 {FeNO} 7 . 49 We propose that the change in the DS ‡ term for the WT cyt P460 reects the degrees of freedom of the His140 ligand. By this hypothesis, the discrepancy between the DS ‡ terms may indicate that the His140 pocket in WT cyt P460 is more rigid, which could decrease NO accessibility on the proximal side of the heme and preclude the NO-dependent His dissociation pathway. Thus, the cyt P460 cross-link "locks" the axial His of the 6c {FeNO} 7 species, thereby slowing its dissociation rate. This impediment allows the oxidation of 6c {FeNO} 7 to {FeNO} 6 to kinetically outcompete protein inactivation.
Our results suggest that the Lys70Tyr cyt P460 mutant can be used as a model for signal transduction by H-NOX proteins. The increased DS ‡ term in the cyt P460 mutant may reect a less rigid pocket surrounding the axial His. In contrast, H-NOX proteins rely on protein conformational changes induced by the formation of the 5c {FeNO} 7 complex to activate signal transduction. Locking the protein conformation would be detrimental to the activation of cyclase activity or interactions with partner signaling proteins. Therefore, the increased degrees of motion in the His pocket may be necessary for signal transduction. To test this hypothesis, mutagenesis studies that perturb the Fe-His interaction will be carried out to explore the effects of His140 pocket alterations on the dissociation of the axial His on other cross-link decient cyt P460 {FeNO} 7 mutants.
Our results also show that the 6c {FeNO} 7 species can be independently generated by treating either Fe III with HNO or Fe II with NO. For the Lys70Tyr cyt P460, these treatments result in either a slow, HNO-concentration-independent His dissociation or a rapid, NO-concentration-dependent His dissociation, respectively. HNO and NO are known to exhibit orthologous physiological effects in humans. 50 The observed differences in the rate laws for the dissociation of axial His from 6c {FeNO} 7 in the presence of HNO or NO may provide insight into the divergent signaling pathways and orthologous physiological effects.
In summary, we have characterized a 6c {FeNO} 7 species on the NH 2 OH oxidation pathway of cyt P460. This species can undergo axial His dissociation to yield an off-pathway 5c {FeNO} 7 species. Kinetic analysis of WT and Lys70Tyr cyt P460 proteins show that the Lys-heme cross-link of the WT protein eliminates a NO-dependent pathway toward 6c-to-5c {FeNO} 7 conversion. Avoidance of this pathway appears to be critical for preserving cyt P460 activity in the periplasmic space of AOB, which necessarily includes NO as an obligate nitrication intermediate. Eyring analyses of the 6c-to-5c {FeNO} 7 conversion were compared to gain insight into how the Lys-heme cross-link increases this activation barrier. Compared with the WT protein pathways, the NO-independent pathway of the cross-link decient mutant has a higher activation entropy. We interpret this observation as evidence that the Lys-heme crosslink confers rigidity to the pocket surrounding the axial His in the WT and propose that this rigidity decreases the number of possible His dissociation pathways. We contend that in addition to obviation of NO-dependent His dissociation, another role for the Lys-heme cross-link is protective: it disfavors 6c-to-5c {FeNO} 7 conversion and consequent inactivation of cyt P460.

Conflicts of interest
There are no conicts to declare.