Nitric-oxide Reductase

We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct νas(Fe-O-Fe) modes at 815 and 833 cm−1 of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm−1mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of nitric-oxide reductase exhibits the ν(Fe3+-NO) and ν(N-O) at 594 and 1904 cm−1, respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to hemeb 3, in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b 3. Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.

The bacterial nitric-oxide reductase (Nor) 1 complex forms the N-N bond during denitrification (1)(2)(3)(4). It is a membrane-bound cytochrome bc complex composed of two subunits, NorC and NorB, that catalyzes the reduction of NO to N 2 O (5). The complex contains four known redox centers: three heme groups and one non-heme Fe atom (5)(6)(7). Heme c (six-coordinate, lowspin) is bound to NorC subunit and functions as the electron entry site of the enzyme. NorB contains one six-coordinate, low-spin heme b and a five-coordinate, high-spin heme b 3 , which, together with a non-heme iron atom, form the dinuclear NO reduction site. The sequence of NorB contains all six histidines that are the ligands of heme a, heme a 3 , and Cu B in cytochrome c oxidase (4).
Resonance Raman (RR) scattering is a powerful technique for the study of heme proteins because the spectra are rich in information about the heme groups (8,9). Moreover, vibrational modes of ligands bound to heme may be assigned by isotopic substitution measurements, and their properties, which reflect ligand structure, may be studied. It has been reported that in oxidized Nor, heme b 3 is not coordinated to the protein by its proximal histidine residue (6). In addition, the single oxygen isotope-sensitive ligand vibration observed at 811 cm Ϫ1 in the RR spectrum was attributed to the as (Fe-O-Fe) of the heme b 3 /non-heme diiron center (7). However, the structural implications involved in the transition from oxidized to reduced enzyme were not reported.
The properties of oxidized Nor, when compared with those of the chemically generated deoxy (five-coordinate) and the nitrosyl complex, can be related to structural changes that take place in the protein upon ligand binding and release. It is these interactions between the heme and the protein that determine the biological properties of the heme protein. Moreover, in unraveling the NO protonation mechanism, it is necessary to establish sites in the enzyme that have exchangeable protons. If there are such sites near the high-spin heme b 3 or the non-heme Fe, then solvent exchange from protonated to deuterated buffers could lead to changes in the RR spectra because the vibrational frequencies are sensitive to effective mass.
In an effort to gain information concerning these observations, we have used 413.1 nm RR excitation to examine the spectra of oxidized Nor upon deuteration and H 2 18 O exchange and compare them to those of the fully reduced form, the NO-bound form, and the NO photoproduct. Our data indicate the presence of two oxygen-sensitive modes at 811 and 833 cm Ϫ1 in the oxidized enzyme. The latter mode also shows deuterium sensitivity. With the identification of the two distinct as (Fe-O-Fe) modes at 815 and 833 cm Ϫ1 of the heme/nonheme diiron center, its structural properties can be described now with more certainty. With the aid of isotopic substitution, we have also characterized the ferric-NO complex by its (Fe 3ϩ -NO) stretching frequency at 594 cm Ϫ1 and (NO) at 1904 cm Ϫ1 , and we postulate that this species is a catalytic intermediate in the NO reduction cycle. In addition, we have shown that the nitrosyl species we detect is photolabile and that the photoproduct has vibrational properties that are different from those of the resting enzyme. Based on our results, we propose a structure-function model of Nor for the transition from the oxidized to the reduced form and from the oxidized to the NO-bound form and its photoproduct.

EXPERIMENTAL PROCEDURES
Nitric-oxide reductase was purified as described elsewhere (5). The activity of the enzyme was measured according to Ref. 5  of Nor was determined using an ⑀ 411 ϭ 3.11 ϫ 10 5 M Ϫ1 ⅐cm Ϫ1 . The pD solutions prepared in D 2 O buffers were measured by using a pH meter and assuming pD ϭ pH (observed) ϩ 0.4. The Raman experiments were carried out with 413.1 nm excitation obtained from a Kr ϩ laser (Coherent K-90). Approximately 40 l of a 50 M enzyme solution in 20 mM Tris, pH 7.4 (or pD ϭ 7.4), was placed in a rotating (4000 -6000 rpm) quartz Raman cell to minimize local heating. The sample cells are custom designed for anaerobic measurements and can be used for recording both the resonance Raman and optical absorption spectra (PerkinElmer Life Sciences Lamda 20 UV-visible spectrometer). The Raman spectra were acquired by using a SPEX 1877 triplemate with an EG&G (model 1530-CUV-1024S) charge-coupled device detector. Frequency shifts in the Raman spectra were calibrated with toluene. The accuracy of the Raman shifts is about Ϯ2 cm Ϫ1 for absolute shifts and about Ϯ1 cm Ϫ1 for relative shifts. The incident laser power was 5-7 milliwatts, and the total accumulation time was 20 -30 min for each spectrum. FTIR spectra were obtained from 200 -300 M samples with a Bruker Equinox 55 FTIR spectrometer equipped with a liquid nitrogen-cooled mercury cadmium telluride detector. Oxidized samples were exposed to 1 atm NO in an anaerobic cell to prepare the nitrosyl adduct and loaded anaerobically into a cell with CaF 2 windows and a 0.025 mm spacer for the FTIR measurements and into a rotating quartz cell for the Raman measurements. NO gas was obtained from Messer, and isotopic NO ( 15 NO) was purchased from Isotec. The FTIR spectrum was obtained as the difference using the buffer as background with 2 cm Ϫ1 spectral resolution and is the average of 1000 scans.

RESULTS
The optical absorption spectrum of oxidized enzyme (Fig. 1, trace A) displays maxima at 411, 530, and 558 nm, which are indicative of low-spin hemes b and c and high-spin heme b 3 . The shoulder at 595 nm is typical of the porphyrin-to-ferric charge transfer transition characteristic of ferric high-spin heme b 3 . The spectrum of the NO-bound oxidized enzyme ( Fig.  1, trace B) was obtained by the direct addition of gaseous NO to the resting enzyme and shows a Soret maximum at 416 nm and the visible transition at 562 nm along with the disappearance of the 595 nm band. For the reduced enzyme ( Fig. 1, trace C) the Soret is at 420 nm, and the visible transitions are at 521, 551, and 558 nm.
The high-frequency RR spectra are used to assess the spin and ligation states of the hemes via the porphyrin marker band frequencies and the status of the proximal and distal environments for the resting, deoxy, NO-bound, and photoproduct form of heme b 3 . The RR spectra of heme proteins in the highfrequency region contain several well-established porphyrin modes termed as the oxidation state 4 or the ligation state ( 3 , 2 , and 10 ) marker modes. Fig. 2 shows the high-frequency RR spectra of oxidized (trace A) and fully reduced (trace B) Nor by using 413.1 nm excitation frequency, in which resonances from all hemes are enhanced, and the resulting assignments are summarized in Table I. The modes of the oxidized enzyme (trace A) at 1373 ( 4 ), 1492 ( 3 ), 1578 ( 2 ), and 1630 ( 10 ) cm Ϫ1 indicate the presence of a five-coordinate high-spin heme b 3 . This suggests that the heme b 3 iron is ligated by either a proximal or a distal ligand (see below). Also present in this spectrum are modes at 1505 ( 3 ), 1584 ( 2 ), and 1639 ( 10 ) cm Ϫ1 , indicating the presence of six-coordinate low-spin heme c and heme b. The mode at 1597 cm Ϫ1 originates from 37 of low-spin heme b or heme c. The 4 bands for the ferric hemes have coincident positions despite the large differences in their redox potentials and porphyrin substituents. This is unusual in view of the 4 dependence upon metal3porphyrin backbonding (d p3 e g ()). In the spectrum of the reduced enzyme (trace B), the 4 mode is located at 1362 cm Ϫ1 , establishing that all hemes are in the ferrous state. The 3 at 1472 cm Ϫ1 , 2 at 1560 cm Ϫ1 , and 10 at 1606 cm Ϫ1 establish the presence of a ferrous, five-coordinate high-spin heme b 3 . The presence of low-spin hemes c and b is shown by 3 intensity at 1494 cm Ϫ1 . The modes at 1584 and 1591 cm Ϫ1 are assigned to 2 of the low-spin hemes b and c, respectively. The 10 vibrational mode for the low-spin hemes is expected around 1620 cm Ϫ1 . Overlap with the vinyl stretching vibration near 1625 cm Ϫ1 , however, hinders the assignment of the 10 vibration. The frequencies of all modes are in agreement with previous analyses of RR spectra of enzymes containing hemes b and c and with those of Fe protoporphyrin model compounds (10,11). Soret band excitation, the RR spectra of ferrous five-coordinate heme b-type with histidine as an axial ligand exhibit ironhistidine stretching modes in the 200 -250 cm Ϫ1 (8). The frequency of the 207 cm Ϫ1 mode (trace D) is similar to that found for the (Fe 2ϩ -His) of other ferrous, five-coordinate high-spin hemes b (8).
In Fig. 4 2 18 O is shown in the difference spectrum (inset, C-B). The observed shift upon deuteration is consistent with hydrogen bonding interactions. The difference spectrum C-B also shows that the 815 cm Ϫ1 band appears to be somewhat broader in D 2 O than in H 2 O. If the bandwidth depends on conformational inhomogeneity, then the 815 cm Ϫ1 band suggests packing of amino acid residues to the bridged oxygen. No other oxygen isotope-sensitive modes are detected in the 160 -900 cm Ϫ1 region, suggesting that neither the symmetric stretch nor the Fe-O-Fe bend is enhanced with 413.1 nm excitation. In addition, we do not detect the 815 and 833 cm Ϫ1 modes in the RR spectrum of the fully reduced enzyme (Fig. 3, trace D), as would be expected from the transition from the oxidized to reduced state. The data demonstrate that both the 815 and 833 cm Ϫ1 modes can be assigned to the as (Fe-O-Fe) of Nor because the 38 -39 cm Ϫ1 shift is in agreement with that expected from the harmonic oscillator approximation for Fe-O-Fe.
The spectral perturbations in the high-frequency region of the RR spectra caused by the addition of NO to the resting enzyme ( Fig. 5 trace A) are limited except for the disappearance of 3 and 2 signals at 1492 and 1578 cm Ϫ1 from the ferric five-coordinate high-spin heme b 3 . Coordination of NO to heme b 3 of the oxidized enzyme shifts the 3 and 2 modes to higher frequency, such that they coincide with those of the low-spin hemes c and b at 1505 and 1584 cm Ϫ1 , respectively (trace B). This indicates that heme b 3 is ferric six-coordinate low-spin in the NO complex. The marked differences we observe in the NO-bound spectra as a function of laser power (trace C) indicate the occurrence of a photolabile species. Identification of 3 and 2 at 1492 and 1572 cm Ϫ1 (trace C) indicates that photodissociation of the NO ligand is producing a five-coordinate high-spin heme b 3 . The core-size band 2 has lost intensity and is downshifted to 1572 cm Ϫ1 , indicating an expanded core in the photoproduct, as compared with the oxidized form. Direct confirmation of the heme b 3 nitrosyl complex requires detection of the heme Fe 3ϩ -NO stretching vibration.  2  1578  1584  1560  1584/1591  1584  3  1492  1505  1472  1494  1505  4  1373  1373  1362  1362  1373  10  1630  1639  1606  1639  11  1550  37  1597  CϭC  1624  1624  The low-frequency RR spectra of the NO-bound form of Nor in the ferric state is shown in Fig. 6. Trace A is from a sample of oxidized enzyme, trace B is from a sample that was exposed to 14 N 16 O, and trace C is from a sample that was exposed to 15 N 16 O. Traces B and C identify the line at 594 cm Ϫ1 for 14 N 16 O as a mode that involves motion of the NO because it downshifts by 6 cm Ϫ1 to 588 cm Ϫ1 upon isotope substitution. Although the ferric-NO species has a frequency that is similar to the 594 cm Ϫ1 observed for non-heme Fe-NO complexes (12), we nevertheless favor a heme b 3 -NO structure for the species we detect. The basis for this lies in the high-frequency RR data. In analogy to other heme proteins, such as Mb and Hb, in which histidine is coordinated to the iron, and the strongest NO isotope-sensitive line present in the spectrum is the Fe-NO stretching mode, we assign the mode at 594 cm Ϫ1 as the Fe-NO stretching mode. The (Fe 3ϩ -NO) stretching vibration in nitrosyl Nor is similar to the (Fe 3ϩ -NO) frequency found in nitrosyl-hemoglobin and to that of the low and high pH nitrosyl-myoglobin (13,14). No other isotope-sensitive mode that could be identified as a possible Fe-N-O bending mode is present in the spectrum of Nor. Traces D and E were obtained with relatively high power, and the absence of modes located at 594 and 588 cm Ϫ1 indicates photodissociation of the NO ligand, as it was observed in the high-frequency experiments. The FTIR spectrum displayed in the inset shows a single vibration at 1904 cm Ϫ1 . This frequency is very similar to the (NO) of the acidic form of metMb-NO (15) and that of horseradish peroxidase-NO (13, 15) but is 18 cm Ϫ1 higher than that of the neutral metMb-NO (15,16). No other modes are present in the frequency range 1800 -2000 cm Ϫ1 that could be identified as possible NO modes of the non-heme iron. The assignment of the Fe 3ϩ -NO and NO stretching frequencies in the reaction of Nor with NO provides a clear description of the nitrosyl complex. Moreover, the 800 -900 cm Ϫ1 spectral region of the RR spectra indicate that photodissociation of the NO ligand from the distal site of heme b 3 is not followed by regeneration of the heme Fe-O-Fe bond.

Two Conformations of the Catalytic Site in Oxidized Nor-
The most reasonable assignment for the two oxygen-sensitive modes present in the spectra of oxidized Nor at 815 and 833 cm Ϫ1 is that they arise from two different conformations of a  (7). The Nor species we detect has a frequency that is close to the 818 -829 cm Ϫ1 bands observed for the six-coordinate ferryl complexes (11) and the five coordinate oxoferrylporphyrin -cation radical (11) observed at 818 cm Ϫ1 . Despite the similarity in these frequencies, we nevertheless favor a Fe-O-Fe structure for the species we detect.
We attribute the conformer with the high-frequency of as (Fe-O-Fe) at 833 cm Ϫ1 as one with a strong positive polar interaction (including hydrogen bonding) between the oxygen atom and the distal residues. This way, a rigid (closed) structure is formed, in which the O atom is stabilized by hydrogen bonding. The conformer with the low-frequency of as (Fe-O-Fe) at 815 cm Ϫ1 is assigned as one with an open structure. The I 833 cm Ϫ1/I815 cmϪ1 indicates that the H-bonded conformer is the major species in the resting form of the enzyme. Because we can eliminate an explanation involving the heme b 3 proximal ligation for rationalizing the open versus closed conformation behavior, we consider as an attractive model one that invokes variation in the extent to which formation of a hydrogen bond between the bridged oxo moiety and distal residues, including the axial ligands of non-heme iron and/or a H 2 O molecule, can occur. This indicates that hydrogen bonding to the oxo moiety by distal residues may be the difference between the two conformations of the Fe-O-Fe unit in Nor. It will be interesting to determine whether there are associated variations in the catalytic function between these two forms because the interaction of the non-heme iron with the heme-bound NO intermediates in the dinuclear site could be quite different for these two forms of the enzyme. Additional studies are necessary to determine whether the two forms of the resting enzyme are a reflection of activity regulation.
Properties of the Fe 3ϩ -NO Adduct of Nor- Table II summarizes (Fe 3ϩ -NO), ␦(Fe 3ϩ -NO), and (NO) frequencies for several heme proteins and model compounds. In the histidinecontaining proteins, using Soret excitation RR spectroscopy, only the Fe-NO stretching and Fe-N-O bending vibrations have been detected (13,19,20). This observation was attributed to the little orbital conjugation that exists between the NO group and the heme in the His-Fe-NO complexes. In the cysteinecontaining enzyme P-450Nor, however, in addition to the Fe-NO stretching vibration, the N-O has been detected using Soret excitation (18). Recently, the (NO) of His-Fe 3ϩ -NO heme proteins was reported by applying UV RR excitation (15). We have searched in the 1800 -2000 cm Ϫ1 RR range and found no evidence for such a mode in our Soret excitation data, in agreement with previous observations in other His-Fe-NO complexes. The stretching frequency of Fe 3ϩ -NO in P-450, which has cysteine as the proximal ligand, is about 70 cm Ϫ1 downshifted compared with that of heme proteins possessing histidine as the proximal ligand (15,16,18). Variations in the basicity of the proximal ligand are likely to contribute to the vibrational differences between the cysteine-and histidinecontaining ferric nitrosyl complexes. Considering the strong electron donating effect of the cysteine sulfur in P-450, it may be argued that the Fe(III)-NO bond strength is a sensitive function of the proximal ligand basicity; the vibrational frequency decreases as the proximal ligand becomes electron donating to the Fe(III)-NO center (see below).
The (NO) of free NO is sensitive to its electronic state. Addition of an electron to the radical NO, which has one electron in the * orbital, weakens the N-O bond and yields NO Ϫ , whereas the removal of an electron strengthens the N-O bond and yields NO ϩ . The frequencies of NO Ϫ , NO ⅐ , and NO ϩ are located at 1284, 1876, and 2345 cm Ϫ1 , respectively (21)(22)(23). The frequencies of (NO) in Mb (acidic form), Mb (neutral form), and horseradish peroxidase are at 1910, 1922, and 1903 cm Ϫ1 , respectively, suggesting that the electronic states of bound NO to these heme proteins are close to that of NO ⅐ and that the NO moiety is electron-deficient. As suggested by Tomita et al. (15), the (NO) in a variety of Fe 3ϩ -NO heme proteins, which all have a neutral His as their trans ligand, is electron-deficient, and the effect of donation from the proximal His is small. Accordingly, we suggest that the NO moiety in Nor adopts the resonance structure Fe ϩ ϭ N ϭ O, which is the same as that of the acidic form of metMb-NO ((NO) ϭ 1910 cm Ϫ1 ) rather than the Fe Ϫ N ϵ O ϩ of the neutral form ((NO) ϭ 1922 cm Ϫ1 ). The latter structure in Mb is stabilized by the lone pair electrons on N ⑀ of the distal histidine, which is located adjacent to the O atom of the bound NO, raising (NO). It also appears that the degree of backbonding in the Fe 3ϩ -NO of Nor is the same as that found in nitrosyl-Hb and Mb, and because it has been established that the frequency of the Fe 3ϩ -NO mode is sensitive to the type of the proximal ligand, its frequency is consistent with a neutral imidazole rather than imidazolate coordination, as occurs in peroxidases (15).
The unusual low frequency of the Fe-CO stretching frequency found in Nor has been attributed to a negatively charged distal pocket (6). A negative charge in the dinuclear center of Nor will stabilize the Fe Ϫ N ϵ O ϩ resonance form and raise (NO). Our results, however, suggest that the Fe ϩ ϭ N ϭ O resonance form is stabilized. Taken together, these observations strongly suggest that the negative polarity of the dinuclear center, suggested previously (6), has no direct control on the strength of the Fe-NO and N-O bonds in our case. The present RR and FTIR results provide clear evidence that there do not appear to be any unusual stereochemical influences in the dinuclear site of Nor resulting in the modification of the Fe-NO bonding, and even though two conformers have been detected in the ferric form of the enzyme, NO binds in a single conformation to heme b 3 . If the NO ligand was bound in two distinct conformations, a second pair of (Fe-NO) and (NO) modes should appear in the RR and FTIR spectra. However, neither of these modes was observed. The single conformation detected in Nor-NO is essentially that observed in nitrosyl-Mb and Hb.
Structural Concept of the Catalytic Site-In an effort to understand the structural implications involved in the transition from oxidized to reduced Nor and from oxidized to the NObound form and its photoproduct, we propose a model that is illustrated in Fig. 7. In the oxidized form, the two different conformations of the dinuclear center represent the open and closed structures in which the high-spin heme b 3 is five-coordinate, and the histidine is not the proximal ligand. Two electrons enter the binding site, resulting in the rupture of the Fe-O-Fe bond and the concomitant reduction of the dinuclear center. In this form of the enzyme, histidine occupies the fifth coordination site on heme b 3 , and the N␦ is hydrogen-bonded as in essentially all heme proteins (24).
The data from the ferric and ferrous forms of the enzyme clearly demonstrate a transition from a five-coordinate highspin heme Fe(III)-O-Fe(III) configuration in the oxidized form to a five-coordinate high-spin configuration His-Fe(II) in the reduced form. The small distance (3.5 Å) between the heme Fe and non-heme Fe implies that disposition of aromatic rings (imidazoles) in the distal pocket can cause steric crowding (4). Thus, although individual residues may supply the dominant interaction for the presence of two conformers, multiple effects can exist, and heme pocket reorganization is also likely to happen. We propose that reduction of the dinuclear center causes a conformation change in heme b 3 . This would induce a heme reorientation and a movement of the heme Fe toward the proximal site, resulting in its ligation to the proximal histidine. Thus, the heme Fe/non-heme Fe distance is expected to be larger in the reduced form. In addition, a heme b 3 -non-heme Fe interaction, which is mediated by the position of the non-heme Fe with respect to the NO bound to heme b 3 , may be very important in the physiological mechanism of NO reduction. One of the most critical features in the structure of Nor is the positioning of the metal centers, especially the relative distance between the non-heme Fe and heme b 3 . These metal centers are known to be close to each other, and in all proposed enzymic mechanisms, the NO or its reduction products have been proposed to interact directly with the heme b 3 and the non-heme Fe (6,25). Therefore, any change in the relative positions of these metal centers is of great significance because it modu- lates the reaction kinetics and even determines the possible reaction products. Addition of NO to the resting enzyme causes the rupturing of the Fe-O-Fe bond and the concomitant ligation of the proximal histidine to the heme iron, producing the His-Fe 3ϩ -NO species. It appears that there is a communication linkage between the distal and proximal sites through bond networks. Therefore, a structural change in the distal site upon NO binding can potentially be communicated to the proximal site through a polypeptide backbone. In the NO association process, we cannot exclude the possibility that the non-heme iron is a way-stop for NO on its route to the heme b 3 binding site, causing the rapture of the Fe-O-Fe bond and, concomitantly, the ligation of the proximal His to the Fe heme b 3 . However, we can exclude with certainty the binding of two NO molecules to the ferric dinuclear center and thus conclude that the heme b 3 has higher affinity for NO compared with the non-heme Fe. The photolability of the ferric NO species is problematic because ferricheme complexes have not been amenable to detailed study due to their chemical reactivity. The experimental data presented here, however, show that the ferric-NO species has high photodissociation quantum yield and that achieving complete photodissociation is not difficult. The photodissociated species has a structure in which the proximal histidine is intact, and the photodissociated NO is not bound to the non-heme Fe.
It is important to note that both proposed mechanisms for the NO reduction by Nor involve two NO molecules in the dinuclear center. In a model proposed by Moënne-Loccoz and de Vries (6), the reaction is initiated when the dinuclear center is fully reduced and one molecule of NO binds to each metal center. They postulated that binding of NO to the ferrous heme b 3 results in the dissociation of the proximal histidine. After dimerization, the bound NO molecules are reduced to N 2 O by using the available electrons on both metal centers. The reduction of 2NO to N 2 O leaves the ferric five-coordinate heme b 3 bridged to the non-heme Fe. Upon reduction of the heme b 3 by electron transfer from the other cofactors, the bridging ligand is lost in favor of the original histidine ligand. Alternatively, it has been proposed by Grönberg et al. (25) that the dinuclear site exists in a mixed valence form (heme b 3 3ϩ /non-heme Fe 2ϩ ) before NO binding and that two NO molecules bind sequentially to the non-heme iron, leaving the heme b 3 essentially as a spectator in the catalytic cycle.
The midpoint redox potentials of each of the metal centers in the enzyme have been measured, and from the unexpectedly low midpoint redox potential of heme b 3 (E m ϭ 60 mV), it was suggested that full reduction of the dinuclear center is thermodynamically unfavorable (25). The redox potentials of the other redox centers were reported at 310 mV for Fe(II) heme c, 345 mV for Fe(II) heme b, and 320 mV for the non-heme Fe(II) (25). Thus, it is possible that under physiological conditions, NO activation in Nor occurs with a mixed valence form of the enzyme in which the low-spin hemes b and c and the non-heme Fe are reduced, and heme b 3 is in the oxidized form. This way, a single molecule of NO binds at the heme b 3 , and the addition of two electrons to heme b 3 Fe 3ϩ -NO yields the two electron reduced species Fe 2ϩ ϪN ϭ O Ϫ . A second NO molecule attacks the N atom of the ferrous-NO species to transiently yield hyponitrite (HONNO-) and thus the N-N bond formation. Cleavage of the N-O bond produces the ferric enzyme, N 2 O, and H 2 O. Such a process occurs in P-450Nor (26), where the electrons needed for the reduction of NO are directly transferred from NADH (2NO ϩ NADH ϩ H ϩ 3 N 2 O ϩ H 2 O ϩ NAD ϩ ϩ H 2 O). The initial step in the reduction process is the formation of the Fe 3ϩ -NO complex, and reduction of the ferric nitrosyl complex by NADH yields a transient species that is spontaneously decomposed to the Fe 3ϩ state. In other enzymes, however, such as cytochrome c oxidase, where the binuclear center is easily reduced, it has been shown that binding of two molecules of NO in the heme pocket of the fully reduced enzyme (4e Ϫ ) leads to oxidation of the heme as well as the nearby copper atom (27). This process involves the uptake of two protons and the generation of N 2 O and H 2 O. The molecular mechanism of NO reduction to N 2 O appears to be complex, inasmuch as our results demonstrate the formation of a photolabile nitrosyl heme b 3 species and that NO coordinates only to heme b 3 . Additional studies of NO coordination to mixed valence Nor are necessary to formulate a complete mechanism for the reduction of NO to N 2 O under physiological conditions.
Resonance Raman spectroscopy was used to characterize the coordination structure and properties of Nor. The measurements that we have reported here unequivocally demonstrate the presence of two distinct modes at 815 and 833 cm Ϫ1 that we assigned to open and closed states of the heme b 3 Fe-O-Fe dinuclear center. The observation of the heme b 3 Fe 3ϩ -NO and N-O stretching modes lays the foundation for identifying other NO modes such as those associated with activated NO intermediates and thereby unraveling the catalytic mechanism of this fascinating enzyme. Further characterization of the heme b 3 Fe 3ϩ -NO pho-toproduct should allow for a clear determination of the molecular relaxation pathway of photodissociated Nor.