Accommodation of two diatomic molecules in cytochrome bo₃: insights into NO reductase activity in terminal oxidases

Abstract

Bacterial heme-copper terminal oxidases react quickly with NO to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N₂O. Previously, we characterized the heme a₃-NO complex formed in cytochrome ba₃ from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu_B(I) to form an end-on NO-Cu_B or side-on copper-nitrosyl complex which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo₃ from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N₂O. The EPR and resonance Raman signatures of the heme o₃-NO complex are comparable to those of the a₃-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o₃-NO complex does not produce a Cu_B-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO demonstrate that an [o₃–NO • OC–Cu_B] tertiary complex can form in bo₃ but not in ba₃. We assign these differences to a greater iron-copper distance in the reduced form of bo₃ compared to that of ba₃. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu_B is not an essential step of the reaction mechanism.

The reduction of nitric oxide (NO) to nitrous oxide (N₂O) (eq. 1) is an obligatory step in the bacterial denitrification pathway which converts nitrate to atmospheric nitrogen.

$$2\text{NO}+2{\text{H}}^{+}+2{\text{e}}^{-}\to {\text{N}}_2\text{O}+{\text{H}}_2\text{O}$$ (1)

Denitrifying NO reductases found in many prokaryotes, including symbiotic and pathogenic bacteria, have been shown to provide those microorganisms resistance to the mammalian immune response. For example, Neisseria gonorrhoeae and Neisseria meningitidis, the causative agents of meningococcal disease in humans, depend upon NO reductases to tolerate toxic concentrations of NO (1–3). These integral protein complexes contain a NorB subunit evolutionarily related to subunit I of heme-copper terminal oxidases (4, 5). There are no crystal structures available for NO reductases (NORs) yet, but sequence alignments and hydropathy plots suggest that the six histidine side chains involved in ligating metal cofactors in terminal oxidases are conserved in norB (6). While O₂ reduction in terminal oxidases occurs at a heme-copper dinuclear site, the reduction of NO by NOR takes place at a heme-nonheme diiron center (7–11). Despite the difference in metal composition, several heme-copper terminal oxidases (i.e., ba₃, bo₃, and cbb₃) are capable of catalyzing the reduction of NO albeit with much lower turnover rates compared to NORs (12–14). This low NO reductase activity is unlikely to be the primary function of these enzymes, but studying their interaction with NO can expand our knowledge of NO reduction mechanisms in NOR and, more generally, in dinuclear active centers.

The catalytic mechanism of NO reduction in terminal oxidases is generally considered to be initiated by the binding of NO to the high-spin heme in the fully-reduced enzyme. Subsequent steps are expected to involve Cu_B, either as a coordination site for a second molecule of NO or as an electron donor and electrostatic partner to a heme-hyponitrite complex [Fe(III)-N₂O₂²⁻ •Cu_B(II)] (11, 15–17). Vos and coworkers have monitored the rebinding kinetics of the photolyzed NO in ba₃-NO at room temperature and interpreted the lack of significant subnanosecond NO rebinding to heme a₃ as evidence of the photolyzed NO binding to Cu_B before rebinding to the heme a₃. (18). In our subsequent FTIR photolysis study of ba₃-NO at cryogenic temperature, we observed the formation of a Cu_B-nitrosyl species with an unusual N-O stretching frequency suggestive of an O-bound (η¹-O) or side-on (η²-NO) configuration (17). The characterization of this complex suggests that the N-N bond formation in ba₃ does not proceed from a transient [a₃-NO • ON-Cu_B] trans ba₃-(NO)₂ complex as proposed by Ohta and coworkers (15). Indeed, if an N-bound Cu_B-nitrosyl was the species formed in the transient ba₃-(NO)₂ complex, one would expect the photoinduced Cu_B-nitrosyl complex to adopt this geometry even in the absence of the heme a₃-NO species nearby.

In light of these results, it is tempting to speculate that the photoinduced Cu_B-nitrosyl species generated in ba₃-NO describes the mode of binding of the second NO molecule. To determine whether the hypothesis drawn from the results with ba₃ applies to other terminal oxidases with NO reductase activity, we now direct our work to the bo₃ quinol oxidase from Escherichia coli. A few investigations have been focused on the interaction of NO with bo₃ from E. coli. Sarti and coworkers measured a low but significant NO reductase activity in bo₃ under reducing conditions (14). On the basis of EPR data, Thomson and coworkers have proposed that two NO molecules bind to Cu_B(II) in the oxidized form of bo₃ (19). To our knowledge, cytochrome bo₃ is the only quinol oxidase reported to exhibit NO reductase activity, and as such, it provides a relevant model for the quinol NOR (qNOR) from N. gonorrhoeae and Neisseria meningitidis (3).

Here we report cryogenic FTIR photolysis experiments on fully reduced bo₃-NO and the mixed gas bo₃-CO/NO complexes. Amperometric measurements of NO concentrations and monitoring of N₂O production by FTIR spectroscopy demonstrate that ba₃ and bo₃ catalyze the same reaction at similar rates. However, the Cu_B-nitrosyl species observed in ba₃-NO does not form in bo₃-NO. Instead, the photolyzed NO of the latter docks at a protein pocket that leads to efficient NO geminate recombination similar to that in ferrous myoglobin-NO (20). FTIR experiments, carried out on ba₃ and bo₃ exposed to NO/CO mixed gas, show concomitant binding of two diatomic molecules only in the dinuclear site of bo₃ to form a [o₃-NO • OC-Cu_B] tertiary complex. The relevance of this [o₃-NO • OC-Cu_B] state to the NO reductase activity in cytochrome bo₃ is discussed in the context of other terminal oxidases and of denitrifying NO reductases.

Materials and methods

Protein stock solutions

The expression and purification of aa₃, ba₃ and bo₃ were performed as previously described (21, 22). For all experiments, cytochrome aa₃ and ba₃ were in 50 mM potassium phosphate pH 7.4 and 0.1% dodecyl β-D-maltoside, and 20 mM Tris-HCl pH 7.5 with 0.05% dodecyl β-D-maltoside, respectively. Cytochrome bo₃ was in 50 mM potassium phosphate pH 8.0 with 0.1% dodecyl β-D-maltoside, 10 mM EDTA, and 5% glycerol.

NO reductase activity measurements

NO stock solutions were prepared by bubbling of NO gas, previously treated with 1 M KOH, into double-distilled water in an anaerobically-sealed vessel for ~15 min at 25°C. The concentration of NO in the solution was determined to be 1.5 mM by titration against ferrous Mb. NO reduction measurements were carried out with a Clark-type NO electrode equipped with a 2 ml gas-tight sample chamber at 20°C in a glove box containing less than 1ppm O₂ (Omnilab System, Vacuum Atmospheres Company). The current was stabilized with a buffer solution containing 10 mM ascorbate and 0.1 mM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) in the sample chamber, followed by three successive additions of saturated NO solution to reach final NO concentrations of 40 to 60 μM. After stabilization of the NO solution, the reduced enzyme was added to reach a final concentration of 7 μM. The current was monitored until it returned to zero.

N₂O production measurements

The production of N₂O by the two terminal oxidases was monitored using the ν(NNO) stretch at 2230 cm⁻¹ in the FTIR spectra (23). Protein solutions were made anaerobic by prolonged purging with argon on a Schlenk line and brought to a final enzyme concentration of 50 μM with 10 mM ascorbate and 0.1 mM TMPD in the glove box. A diethylamine NONOate (Cayman Chemical, Ann Arbor, MI) stock solution, in 0.01 M NaOH, was prepared based on its ε_{250 nm} = 9180 M⁻¹ cm⁻¹ extinction coefficient and an aliquot was used to confirm the concentration of the NO produced by monitoring the conversion of deoxymyoglobin to the nitrosyl complex. Quickly after the addition of NONOate to the protein solution, a 33-μL droplet of sample was deposited on a CaF₂ window and a second CaF₂ window was dropped on the sample. The optical pathlength was controlled by a 100-μm Teflon spacer. The FTIR cell was placed in the sample compartment of the FTIR instrument. FTIR spectra were obtained on a Perkin-Elmer system 2000 equipped with a liquid-N₂-cooled MCT detector and purged with compressed air, dried, and depleted of CO₂ (Purge gas generator, puregas LLC). Sets of 100 scans accumulations were acquired every 2 min, at a 4-cm⁻¹ resolution, until no further growth of the N₂O IR band was observed. These data were compared to a calibration curve obtained from solutions with varying N₂O concentrations.

EPR experiments

EPR spectra were obtained on a Bruker E500 X-band EPR spectrometer equipped with a superX microwave bridge and a dual mode cavity with a helium flow cryostat (ESR900, Oxford Instruments, Inc.). The microwave power, modulation amplitude, magnetic field sweep, and the sample temperature were varied to optimize the detection of all potential EPR active species before and after illumination of the nitrosyl complexes.

RR experiments

Typical enzyme concentrations used were 150 μM. The RR spectra were obtained using a custom McPherson 2061/207 spectrograph (set at 0.67 m with variable gratings) equipped with a Princeton Instruments liquid-N₂-cooled CCD detector (LN-1100PB). Kaiser Optical supernotch filters were used to attenuate Rayleigh scattering generated by the 413-nm excitation of an Innova 302 krypton laser (Coherent, Santa Clara CA). Spectra were collected using a 90° scattering geometry on room-temperature samples mounted on a reciprocating translation stage. Frequencies were calibrated relative to indene and aspirin standards and are accurate to ±1 cm⁻¹. Polarization conditions were optimized using CCl₄. The integrity of the RR samples, before and after illumination, was confirmed by direct monitoring of their UV-vis spectra in Raman capillaries.

FTIR photolysis experiments

FTIR photolysis experiments were carried out as previously described (17). After prolonged purging with argon on a Schlenk line, the sample was fully reduced by addition of 10 mM dithionite in an anaerobic glove box. NO gas (¹⁴NO from Airgas and Aldrich, ¹⁵NO from ICON), initially treated with 1 M KOH solution, was added to the sample headspace to achieve an NO partial pressure of 0.1 atm. After 1 min of incubation at room temperature, 15 μL of the protein solution was deposited as a droplet on a CaF₂ window and a second CaF₂ window was dropped on the sample using a 15-μm Teflon spacer to complete the FTIR cell.

CO/NO mixed-gas experiments were carried out on ~350 μM reduced-protein solutions containing 10 mM ascorbate, 0.1 mM TMPD, and 25% glycerol. The sample headspace was thoroughly exchanged with pure CO gas to reach saturation (¹²CO purchased from Airgas or ¹³CO purchased from ICON) and incubated for 15 min at room temperature. A few μL of a stock solution of NONOate was added to the sample to produced 3.0 equiv NO. Immediately after the addition of NONOate, a 15-μL droplet of sample was deposited on a CaF₂ window with a 15-μm Teflon spacer, and a second CaF₂ window was dropped on the sample to complete the IR cell. The 25% glycerol content of the sample insured minimal degassing of CO during this procedure. Alternatively, the reduced protein (with the same concentration and buffer conditions as above) was exposed to a 0.1-atm NO partial pressure for 1 min and the headspace replaced with pure CO gas and incubated for 10 min at room temperature before transferring the sample to the IR cell.

For all samples, once the IR cell was securely sealed, the presence of the desired complexes was confirmed by obtaining a UV-vis spectrum of the sample using a Cary 50 spectrophotometer (Varian). The FTIR cell was then mounted to a closed-cycle cryogenic system (Displex, Advanced Research Systems) and installed in the sample compartment of the FTIR instrument to keep in the dark while the temperature dropped to 30 K. The temperature of the sample was monitored and controlled with a Cry-Con 32 unit. Sets of 1000-scan accumulations were acquired at a 4 cm⁻¹ resolution by the Perkin-Elmer system 2000. Photolysis of the nitrosyl and carbonyl complexes was achieved with continuous illumination of the sample directly in the FTIR sample chamber with a 50 W tungsten lamp after filtering out heat and NIR emission. This same illumination procedure was used to follow the dissociation and rebinding processes by UV-vis absorption spectroscopy with a Cary-50 spectrometer.

The reversibility of the photolysis events studied here was confirmed by comparing successive ‘dark’ and ‘illuminated’ UV-vis absorption spectra, and ‘dark’ minus ‘illuminated’ FTIR difference spectra obtained at 30 K after raising the temperature of the sample. A first evaluation of the temperature dependence of the rebinding process was obtained by UV-vis absorption, raising the sample temperature incrementally by 10 K until the return of the ‘dark’ spectrum was observed. The comparison of a first ‘dark’ minus ‘illuminated’ FTIR difference spectrum obtained at 30 K with a second 30-K difference spectra obtained after an incubation period at higher temperature, referred to below as annealing temperature, provides a reliable means to compare rebinding-temperatures between distinct photolabile species.

Results

NO reductase activity measurements

While both ba₃ and bo₃ have been reported to possess NO reductase activity (12, 14), their steady-state turnover rates have not been compared in side-by-side experiments. Thus, we carried out parallel NO reductase activity measurements on ba₃ and bo₃ by monitoring NO consumption amperometrically under reducing conditions (10 mM ascorbate and 0.1 mM TMPD) (Figure 1). Upon addition of myoglobin, aa₃, ba₃, and bo₃ to the NO solutions, a rapid initial decay of current is assigned to the stoichiometric binding of NO to ferrous high-spin hemes. While no further current change was observed with myoglobin and aa₃, ba₃ and bo₃ displayed NO consumption with initial rates of 3.4 mol NO/mol ba₃-min and 2.6 mol NO/mol bo₃-min, respectively, at [NO] = 40 μM (Figure 1). These values match that previously reported by Giuffre et al. (3.0 ± 0.7 mol NO/mol ba₃-min at [NO] = 55 μM) (12) and complement the measurement by Butler et al. (0.3 mol NO/mol bo₃-min at [NO] = 5 μM) (14). It is worth noting that, in the presence of excess reducing agent, both enzymes remained as mononitrosyl complexes after the turnover measurements (data not shown). This observation indicates that, in both enzymes, the binding affinity for the second NO is much lower than that of the first NO.

Figure 1.

NO binding and reductase activity of ferrous myoglobin (A), aa₃ (B), ba₃ (C), and bo₃ (D) at 25°C. Black arrows show time points where each enzyme were added to react a final concentration of 7 μM. For ba₃ and bo₃, the early part of the trace is also shown expanded 10-times to reveal the initial NO-binding steps.

The production of N₂O was monitored by the FTIR measurement of the antisymmetric N-N-O stretch mode ν₃ of N₂O at 2231 cm⁻¹. Using calibration curves with N₂O-saturated solutions, the 2231-cm⁻¹ absorption values of the ba₃ and bo₃ solutions confirm that all of the NO consumed is converted to N₂O. (Figure S1). As expected, N₂O was not detected in FTIR measurements when the terminal oxidases were replaced by myoglobin (data not shown).

UV-vis, EPR and RR characterization of bo₃-NO

Exposure of fully-reduced bo₃ to a headspace containing 0.1 atm NO results in the rapid formation of bo₃-NO. The Fe(II) heme-o₃ Soret absorption at 426 nm is blue-shifted to generate a new Soret band at 416 nm, and there are only minor changes in the visible range of the room-temperature absorption spectra (Figure 2, inset). The Soret absorption at 416 nm, assigned to the heme-o₃-NO complex, is also observed at 30 K, but disappears following illumination (Figure 2). As reported previously (24), the EPR spectrum of the bo₃-NO complex is characteristic of a 6-coordinate low-spin heme iron(II)-nitrosyl species with g values centered around 2 (2.102, 2.01, 1.99) and nine-line ¹⁴N-hyperfine structure (A_NO = 20 G, A_His = 6 G) equivalent to signals observed in ba₃-NO (Figure S2). In addition, this EPR spectrum includes an easily saturated, isotropic g = 2.005 with a 10 G linewidth which was previously assigned to a semi-quinone radical (25, 26). Unlike the EPR features associated with the heme o₃-NO, this signal is insensitive to short illumination at cryogenic temperatures (Figure S2). The only new EPR signal observed after illumination is that of free NO at g = 1.97, which is best observed at high microwave power and below 10 K (data not shown).

Figure 2.

UV-vis spectra in dark (black) and illuminated (red) and dark minus illuminated difference spectra (blue) of bo₃-NO complex at 30 K. The inset shows the room temperature UV-vis spectra of reduced bo₃ (red) and the bo₃-NO complex (black).

RR spectra of the bo₃-NO complex, obtained with a 413-nm excitation at room temperature, display enhancement of vibrational modes from the heme o₃-NO complex (Figure 3). The porphyrin skeletal modes ν₄, ν₃, ν₂, and ν₁₀ at 1361, 1504, 1587, and 1638 cm⁻¹, respectively, are characteristic of a 6-coordinate low-spin heme-nitrosyl species. Vibrational modes involving the Fe-N-O unit are indentified by their ¹⁵N¹⁸O-downshifts (Figure 3). The ν(N-O)o₃ mode is observed at 1615 cm⁻¹ and exhibits a 67-cm⁻¹ downshift with ¹⁵N¹⁸O that is within 5-cm⁻¹ of the calculated shift for a diatomic N-O oscillator. Two bands, at 534 (−17) and 440 (−13) cm⁻¹, are assigned to ν(Fe-NO) and δ(Fe-NO) modes, respectively, even though significant mixing exists between these two modes (27–29). The unusually intense d(Fe-NO) band, which is not observed in ba₃-NO (17, 30), and the low ν(Fe-NO) frequency suggest that the Fe-N-O angle is smaller than the 140° equilibrium value. The low ν(N-O)o₃ frequency is also consistent with a small Fe-N-O angle which favors the Fe(III)NO⁻ resonance structure and N-O double-bond character.

Figure 3.

Low- and high-frequency RR spectra of bo₃-¹⁴N¹⁶O (red), bo₃-¹⁵N¹⁸O (blue), reduced bo₃ (black), and the bo₃-¹⁴N¹⁶O minus bo₃-¹⁵N¹⁸O difference spectrum (green) obtained with a 413-nm excitation at room temperature (protein concentration ~150 μM).

Low-temperature FTIR photolysis of the bo₃-NO and bo₃-(NO)(CO) complexes

Previously, we used photolysis experiments with ba₃-NO at 30 K to isolate ν(N-O) vibrations in ‘dark’ minus ‘illuminated’ FTIR difference spectra (17). These experiments revealed that the disappearance of the ν(N-O)a₃ at 1622 cm⁻¹, consistent with the dissociation of NO from the heme and accompanied by the formation of a Cu_B-nitrosyl complex with a negative ν(N-O)Cu_B at 1589 cm⁻¹. In the case of bo₃-NO, the FTIR ‘dark’ minus ‘illuminated’ difference spectra show a sharp positive band at 1610 cm⁻¹ that is readily assigned to the ν(N-O)o₃ by its 30-cm⁻¹ downshift with ¹⁵NO, but there are no negative signals suggestive of a Cu_B-nitrosyl species (Figure 4). Instead, a negative band at 1863 cm⁻¹ that shifts -34 cm⁻¹ with ¹⁵NO is characteristic of a ν(N-O) from an NO molecule docked in a proteinaceous pocket, as observed with the nitrosyl complex of myoglobin (20). Varying buffer and salt conditions had no effect on the FTIR difference spectra of bo₃-NO (Figure S3). Thus, these experiments suggest that, despite the structural similarities of the heme-copper sites in these terminal oxidases (31, 32) and their efficient capture of photolyzed CO by Cu_B(I) in both terminal oxidases, NO transfer from heme to Cu_B does not occur in bo₃. This interpretation is also supported by a difference between bo₃-NO and ba₃-NO in their temperature dependence for geminate rebinding of NO. Indeed, after a first illumination at 30 K, the photolyzed ba₃-NO complex must be annealed to 90 K to recover the full amplitude of the ‘dark’ minus ‘illuminated’ FTIR difference spectrum (17), whereas the temperature only needs to be raised to 60 K to allow complete rebinding of NO to heme o₃ in bo₃-NO (data not shown).

Figure 4.

FTIR difference spectra (‘dark’ minus ‘illuminated’) of bo₃-NO (top traces) and ba₃-NO (bottom traces) at 30 K. The spectra were obtained with protein concentration near 350 μM and were normalized based on the room temperature UV-vis spectra obtained directly from the FTIR cell (15 μm pathlength).

To gain further insight into the catalytically-relevant step in which two NO molecules interact with the heme-copper, and anticipating the formation of a non-reactive [(heme-copper)(NO)(CO)] tertiary complex, we carried out experiments on reduced ba₃ and bo₃ proteins with consecutive exposure of to CO and NO gases. These experiments succeeded in forming a [o₃-NO • OC-Cu_B] complex in bo₃. In ba₃, exposure of ba₃-CO, in the presence of excess CO, to 3 equiv of NO minutes before freezing (see experimental section) resulted in the formation of ba₃-NO and ba₃-CO complexes that were easily distinguishable in the FTIR spectra (Figure 5). While rebinding of the photolyzed CO required annealing the sample above 220 K, the rebinding of NO was complete after annealing at 100 K. This difference in rebinding temperature allows the separation of FTIR features associated with ba₃-NO and ba₃-CO. When the same experiment was carried out with bo₃, features in the FTIR difference spectra associated with bo₃-NO and bo₃-CO complexes were also observed; however, the NO dissociation process induces a differential signal in the ν(C-O)Cu_B region that shifts –47 cm⁻¹ with ¹³CO (Figure 5). We assign this signal, centered at 2057 cm⁻¹, to a perturbation of the Cu_B-carbonyl as NO is dissociated from heme o₃. The ν(NO)o₃ in these mixed-gas experiments is observed at 1610 cm⁻¹ and is indistinguishable from the ν(NO)o₃ observed when the complex is formed with pure NO. The relative intensities of the ν(N-O)o₃ and ν(C-O)o₃ bands suggest that the active site in the bo₃-CO state represents only 10% of the sample, while the remaining 90% contains the heme o₃-NO complex. Furthermore, on the basis of the ν(C-O)Cu_B band observed in the ‘dark’ spectrum (Figure 6), we estimate that out of the 90% of nitrosylated active site, at least 10% binds CO to form the [o₃-NO • OC-Cu_B] complex. Thus, although the active site of bo₃ cannot bind two CO molecules at the same time, binding of NO to heme a₃ allows the subsequent binding of CO to Cu_B. The ν(C-O)Cu_B band in the [o₃-NO • OC-Cu_B] complex is symmetric and centered at 2057 cm⁻¹, which contrasts the multiple conformers observed in the light-induced [o₃ • OC-Cu_B] state (Figure 6). Changing the order of gas exposure by forming the heme-nitrosyl complexes before the addition of excess CO had no effect on the FTIR data obtained with bo₃ and ba₃ (data not shown).

Figure 5.

FTIR difference spectra (‘dark’ minus ‘illuminated’) of ba₃-CO/NO (top) and bo₃-CO/NO (bottom), before (black) and after annealing at 120 K (blue). Also shown for comparison, are the ‘dark’ minus ‘illuminated’ difference spectra for the pure-CO complexes (red traces) and the bo₃-¹³CO/NO difference spectra after illumination and annealing at 120 (pink). The spectra were obtained with protein concentration near 350 μM and were normalized based on the room temperature UV-vis spectra obtained directly from the FTIR cell (15 μm pathlength).

Figure 6.

Comparison of FTIR difference spectra in the range of the ν(¹²C-O)Cu_B modes in bo₃. From top to bottom: ¹²CO minus ¹³CO difference spectrum from the ‘dark’ bo₃-CO/NO (black), ¹⁴NO minus ¹⁵NO difference spectrum from the ‘dark’ bo₃-NO (red), and ‘illuminated’ minus ‘dark’ bo₃-¹²CO (bottom, black). The spectra were obtained with protein concentration near 350 μM and were normalized based on the room temperature UV-vis spectra obtained directly from the FTIR cell (15 μm pathlength).

Discussion

Numerous studies with fully-reduced terminal oxidases have shown that the five-coordinated ferrous high-spin heme efficiently binds NO to form a stable ferrous low-spin nitrosyl complex (33–38). Several terminal oxidases, including T. theromophilus ba₃ and E. coli bo₃, have been shown to further react with a second NO molecule to catalyze the reduction of NO to N₂O (12, 14). Recently, we reported the formation of a photoinduced Cu_B-nitrosyl species with an end-on NO-Cu_B or a side-on Cu_B-nitrosyl configuration in ba₃ at cryogenic temperature (17) and proposed that this species reflects the binding geometry of the second NO molecule involved in the NO reductase reaction catalyzed by ba₃ (17). In the present work, we show that the bo₃ quinol oxidase from E. coli reduces NO at a rate equivalent to that of ba₃ (~3 mol NO/[E] mol-min at [NO] = 40 μM). In analogy to ba₃, a stable 6-coordinate low-spin heme-o₃ nitrosyl complex is observed, which exhibits Fe-N-O stretching frequencies suggestive of a bent Fe-N-O geometry (27, 28). Illumination of bo₃-NO at 30 K dissociates the heme-nitrosyl complex with equivalent efficiency as in ba₃-NO, but complete rebinding of NO to the heme o₃ occurs after annealing the sample to 60 K, which is significantly lower than the 90-K annealing temperature measured in ba₃-NO (17). Thus, the photolyzed bo₃-NO state is thermodynamically less favored than the corresponding nitrosyl complex in ba₃. Comparison of low-temperature FTIR photolysis data for bo₃-NO and ba₃-NO support this conclusion. Indeed, light-induced FTIR difference spectra show that stabilization of the photolyzed NO through interactions with Cu_B(I) does not occur in bo₃-NO as it does in ba₃-NO. Rather, the photolyzed FTIR spectra of bo₃-NO reveals a ν(N-O) band at 1863 cm⁻¹ which corresponds to an NO molecule docked in a proteinaceous pocket.

What prevents the formation of a light-induced Cu_B-nitrosyl in bo₃? Because the photolysis process with the bo₃-CO complex leads to the efficient capture of the photolyzed CO by Cu_B, a lack of an open coordination site on Cu_B can be ruled out. Extensive RR and FTIR studies of the heme-copper carbonyl complexes have revealed different configurations, which have been named α, β, and γ forms that correspond to increasing levels of steric restrictions at the active site pocket (39–42). According to these studies, the ba₃-CO complex represents a highly-restricted site (γ form) (41), while the bo₃-CO complex offers more open configurations of the dinuclear site (40). The iron-copper distances reported for the crystal structures of terminal oxidases concur with this view, with metal-metal distance ranging for 5.3 Å in bo₃ and 4.4 Å in ba₃ (31, 32, 43–46). However, metal-metal distance comparison from crystal structures should be used with caution since the structure of bo₃ was only solved at 3.5 Å resolution, and because the redox state of the active sites during the X-ray diffraction data acquisition is not always clearly defined. This point is exemplified by a recent x-ray crystallographic study of a surface double mutant of cytochrome ba₃ (E4Q & K258R) (47) which shows that the iron-copper distance can vary from 4.7 Å in the X-ray photoreduced oxidized crystal to 5.05 Å in chemically reduced crystals (48). However, the FTIR analysis of this double mutant of cytochrome ba₃ in the -CO, -NO, and -NO complex in presence of CO gas produced identical results to that of wild type ba₃ (Figures S4 and S5).

Based on our FTIR results, we hypothesize that in bo₃-NO, a larger metal-metal distance prevents the efficient capture of the photolyzed NO by Cu_B. This hypothesis also explains the formation of a bo₃-(NO)(CO) complex which is not observed in ba₃. This [o₃-NO • OC-Cu_B] state is evidenced by a differential signal centered at 2057 cm⁻¹ from a Cu_B-carbonyl complex perturbed by the photolysis of NO from heme-o₃. It is striking that one CO and one NO can co-exist at the dinuclear site of bo₃, while two CO molecules cannot.(49) This observation suggests that the distance between heme o₃ and Cu_B is just large enough to accommodate one CO at the Cu_B with one NO at the heme o₃ in a bent geometry but not large enough to accommodate two linear diatomics. The limited accumulation of [o₃-NO • OC-Cu_B]) to ~12% of the active sites may reflect the presence of multiple conformations of the heme-copper site (in bo₃). The ν(N-O)o₃ frequency in the [o₃-NO • OC-Cu_B] state is equivalent to that observed in the [o₃-NO • Cu_B] state. However, the [Fe-NO • OC-Cu_B] state exhibits only one ν(C-O)Cu_B at 2057 cm⁻¹, which contrasts with the multiple ν(C-O)Cu_B bands observed at 2058 and 2073 cm⁻¹ observed after photolysis of bo₃-CO at low temperature (Figure S4) (50, 51). EXAFS measurements suggest that high ν(C-O)Cu_B frequencies could correspond to active site populations where one of the three coordinating histidines to Cu_B is weakly bound (52). Regardless of the structural significance of the different ν(C-O)Cu_B frequencies, the mixed-gas experiments suggest that the conformer that correspond to the high ν(C-O)Cu_B does not bind CO in presence of the heme o₃-NO complex.

Reports of concomitant binding of two diatomic molecules at heme-copper dinuclear sites are scarce. In fully-reduced bovine and prokaryotic aa₃, the loss or alteration of the EPR signal from a₃-NO at high NO concentration has been assigned to the binding of a second NO molecule to Cu_B (35, 53). Using FTIR spectroscopy, Caughey and coworkers observed two ν(NO)s in bovine aa₃: one at 1610 cm⁻¹ assigned to a₃-NO, and one at 1700 cm⁻¹ assigned to Cu_B-NO (54). Presumably, the lack of NO reductase activity in aa₃ terminal oxidases permits the accumulation of a stable [{FeNO}⁷ • {CuNO}¹¹] complex, but the presence of two NO molecules in the active site of ba₃ and bo₃ is expected to represent a highly reactive intermediate within the NO reductase turnover. Although the characterization of a [o₃-NO • OC-Cu_B] complex in bo₃ suggests that this active site can also accommodate a [{FeNO}⁷ • {CuNO}¹¹] trans-complex, this state may also correspond to a dead-end adduct as in the aa₃ systems.

Low-temperature photolysis experiments are a sensitive probe of dinuclear heme-copper active site. The results presented here show that fully-reduced ba₃ and bo₃ bind a first NO molecule to the high-spin heme-iron(II) in similar fashion, but the distance of the Cu_B site relative to the heme iron differs significantly in the two proteins. In ba₃, the close vicinity of the heme a₃ and the Cu_B allows for the transfer of the photolyzed NO from the heme to Cu_B in a side-on geometry (17). In bo₃, the larger metal-metal distance does not restrict the coordination of a second diatomic molecule at the Cu_B site (as long as it can adopt a bent geometry). Our experiments do not determine whether the Cu_B(I) site in bo₃-NO binds a second NO molecule to form a trans [{FeNO}⁷/{CuNO}¹¹] complex. Nevertheless, the comparison of ba₃ and bo₃ shows that the mechanism of NO reduction can accommodate the difference in heme-copper distances in these two active sites. This conclusion argues against the coordination of the second NO molecule to Cu_B(I) as an essential step in the reaction mechanism. Instead, the role of the Cu_B site may be limited to promote the formation of a heme iron-hyponitrite species through electrostatic interactions (11, 16).

Supplementary Material

1_si_001. Supporting Information available.

FTIR spectra monitoring N₂O production, EPR spectra of the bo₃-NO complex, FTIR spectra of bo₃-NO in different pH and salt concentration, and comparison of FTIR data for ba₃ wild type and double mutant E4Q/K258R. This material is available free of charge via Internet at http://pubs.acs.org.