J. Phys. Chem. B 2008, 112, 1851-1857
1851
Resonance Raman Spectroscopy of Nitric Oxide Reductase and cbb3 Heme-Copper Oxidase Eftychia Pinakoulaki† and Constantinos Varotsis*,‡ Department of Chemistry, UniVersity of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus, and Department of Chemistry, UniVersity of Crete, 71003 Voutes, Heraklion, Crete, Greece ReceiVed: September 11, 2007; In Final Form: NoVember 11, 2007
Elucidating the structure and properties of the active sites in cbb3 heme-copper oxidase and in nitric oxide reductase (Nor) is crucial in understanding the reaction mechanisms of oxygen and nitric oxide reduction by both enzymes. In the work here, we have applied resonance Raman (RR) spectroscopy to investigate the structure and properties of the binuclear heme b3-CuB center of cbb3 heme-copper oxidase from Pseudomonas stutzeri and the dinuclear heme b3-FeB center of Nor from Paracoccus denitrificans in the ligand-free and CO-bound forms and in the reactions with O2 and NO. The RR data demonstrate that in the Nor/NO reaction, the formation of the N-N bond occurs with the His-Fe heme b3 bond intact, and reformation of the heme b3-O-FeB dinuclear center causes the rupture of the proximal His-Fe heme b3 bond. In the reactions of Nor and cbb3 with O2, distinct oxidized heme b3 species, which differ from the as-isolated oxidized forms, have been characterized. The activation and reduction of O2 and NO by cbb3 oxidase and nitric oxide reductase are compared and discussed.
Introduction Considerable progress has been made over the past few years in understanding the details of the molecular structure and function of heme-copper oxidases.1-7 The mechanisms of O2 reduction and proton translocation have attracted attention, as they are critical in understanding the molecular operation of this class of enzymes. On the basis of the structural similarities between the Pseudomonas stutzeri NO reductase and the terminal cbb3 oxidase, it has been proposed that a common phylogeny of aerobic respiration and bacterial denitrification exists.8 On the same basis, it has been also suggested that the essential difference between the two classes of enzymes lies in the organization of their active site. In the denitrification process, nitric oxide reductase (Nor) catalyzes the two-electron reduction of NO to N2O through a heme b3-non-heme FeB dinuclear center, whereas in respiration cytochrome c oxidase catalyzes the reduction of O2 to H2O in a binuclear heme a3-CuB center.1-20 Several themes have emerged in the studies of biological control of dioxygen and nitric-oxide chemistry.1,12 The most commonly encountered metals in these processes are iron and copper in mononuclear, binuclear, and dinuclear centers. Apparently, evolution has clearly selected the means by which to take advantage of the unique O2 and NO chemistry; several enzymes have evolved that are able to activate both O2 and NO kinetically and control their redox chemistry.1,12,15 Recent studies have indicated that ba3- and caa3-type heme-copper oxidases isolated from Thermus thermophilus display a low NO reductase activity.8 On the other hand, cbb3 oxidase from Pseudomonas stutzeri has the highest NO reductase activity (Km(NO) ) 12 µM) among any known heme-copper oxidases indicating a closer phylogenic relationship between cbb3 and Nor.21 The Km(NO) of cbb3 isolated from P. stutzeri is 4 orders of magnitude greater than that of Nor purified from the same * To whom correspondence should be addressed. Tel: +30-2810-545053. Fax: +30-2810-545001. E-mail:
[email protected]. † University of Cyprus. ‡ University of Crete.
organism.12,21 It has also been reported that in Nor, the maximal turnover number with oxygen (120 electrons s-1) is in the same order of magnitude as the maximal turnover observed with NO.16 Nor contains one c- and one b-type low-spin heme and a five-coordinate, high-spin heme b3 which together with a nonheme iron atom form the dinuclear NO reduction site.12-20 Resonance Raman (RR) spectroscopy has revealed the properties of the dinuclear center of oxidized, reduced, and NO-bound Nor from Paracoccus denitrificans.17-19 The spectra of the oxidized enzyme show two distinct νas(Fe-O-Fe) modes at 813 and 833 cm-1 that have been attributed to two different conformations (open and closed) of the catalytic site of the enzyme.19 Moreover, the ferric nitrosyl complex of Nor has been characterized by its ν(Fe3+-NO) and ν(N-O) at 594 and 1904 cm-1, respectively.19 Comparison of the cbb3 oxidase to Nor may provide the means to identify conserved structural features, which can be assumed to be involved in basic functions common to both classes of enzymes. In contrast, dissimilarities between these enzymes are likely to be involved in the fine-tuning to specific needs demanded by differences in their local biological environments. The cbb3 oxidase isolated from P. stutzeri contains three c-type low-spin hemes, one low-spin b-type heme, and a heme b3-CuB binuclear center.22-23 Time-resolved step-scan Fourier transform infrared (FTIR) experiments have demonstrated that in cbb3-cytochrome c oxidase from P. stutzeri the decay of the transient CuB1+-CO complex (ν(CO) ) 2065 cm-1) is concurrent with the formation of the heme b32+-CO complex.24 The ferrous and ferric nitrosyl cbb3 complexes have also been characterized.11,25 Addition of NO to the fully reduced enzyme causes the cleavage of the His-Fe heme b3 bond, producing a five-coordinate heme b32+-NO complex with characteristic ν(Fe-NO) and ν(NO) at 524 and 1679 cm-1, respectively.25 In an effort to understand the similarities in the structure and properties of the active site of the cbb3 oxidase and Nor, we have investigated the RR spectra of ligand-free, CO-bound forms of the reaction products that are formed by the direct mixing of
10.1021/jp077295o CCC: $40.75 © 2008 American Chemical Society Published on Web 01/23/2008
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the CO-bound fully reduced forms of cbb3 and Nor with oxygen and of the reaction product of the fully reduced Nor/NO reaction. The RR data from the fully reduced Nor/NO reaction product illustrate the formation of a 6C/HS (six-coordinate high-spin) heme b3 species with water as the possible sixth ligand. We suggest that the oxygen atom of the coordinated H2O molecule is the source of the regenerated oxidized (as-isolated) His-heme b3-O(H)-FeB species. We conclude that rupturing of the proximal His-Fe heme b3 bond occurs to form the oxidized heme b3-O(H)-FeB dinuclear center. The data also indicate that the formation of the N-N bond in the denitrification process occurs with the His-Fe heme b3 intact and that the dinuclear center becomes fully oxidized during the catalytic cycle. In the Nor/O2 reaction, we have detected and have characterized a 5C/ HS species in which the proximal to heme b3 histidine is intact. On the other hand, in the cbb3/O2 reaction, we have characterized the end product as a 6C/HS heme b3 species in which the ν2 mode is 15 cm-1 higher than that of the as-isolated enzyme. Experimental Methods Nor was purified as described elsewhere.16 The NO-reductase activity of the enzyme was measured according to ref 16 and was approximately 40 µmol/mg/min (46e-/s). The samples were concentrated to 150 µM in 20 mM Tris pH 7.4 containing 0.05% dodecyl β-D-maltoside and were stored in liquid nitrogen until use. The concentration of Nor was determined using an 411 ) 3.11 × 105 M-1 cm-1. The microaerobic cbb3-cytochrome c oxidase was isolated from Pseudomonas stutzeri according to ref 22. The activity of cbb3 was measured in the presence of 2,2,4-trimethyl-1,3-pentanediol (TMPD) and ascorbate as electron donors as described in ref 22, and the rate of oxygen consumption was 180 mol O2 s-1. The resonance Raman spectra were acquired as described elsewhere.9,10 The incident laser power of the 413.1 nm excitation frequency was 5-7 mW, and the total accumulation time was 5-10 min for each spectrum. Dithionite-reduced samples were exposed to 1 atm NO or CO in an anaerobic rotating quartz cell for the Raman measurements. NO and CO gases were obtained from Messer (Germany), and isotopic NO (15NO) was purchased from Isotec. Results The optical absorption spectrum of oxidized Nor (Trace A in Figure 1) displays maxima at 411, 530, and 558 nm, which are indicative of low-spin hemes b and c and high-spin heme b3. The shoulder at 595 nm is typical of the porphyrin-to-ferric charge transfer (CT) transition characteristic of ferric high-spin heme b3. For the reduced enzyme (trace B in Figure 1), the Soret is at 420 nm and the visible transitions are at 521, 551, and 558 nm. NO coordinates to heme proteins in both the ferric and the ferrous oxidation states of the heme iron atom, yielding characteristic spectra. Upon exposure of the reduced enzyme to an atmosphere of NO gas, trace C with a Soret maximum at 415 nm and the visible transitions, albeit with reduced intensities at 521, 551, and 558 nm, is obtained. By allowing the sample to incubate for 10 min, conversion of the low-spin ferrous hemes b and c to their oxidized form is detected, and therefore the enzyme progressively becomes fully oxidized, as is evident in trace D. Removal of NO by flushing with argon gas regenerates the spectrum (trace E) of the fully oxidized enzyme in which the CT band at 595 nm is regenerated. The high-frequency RR spectra of Nor in the oxidized (trace A), reduced (trace B), CO-bound (trace C), and reoxidized form (trace D) are shown in Figure 2. With our excitation wavelength at 413.1 nm, all hemes contribute to the intensity of the Raman
Figure 1. Optical absorption spectra of nitric oxide reductase (Nor) from P. denitrificans in the as-isolated form (trace A), the dithionite reduced form (trace B), the fully reduced/NO reaction (trace C, t ) 3 min), the fully reduced/NO reaction (trace D, t ) 10 min), and after the removal of excess NO by flashing with argon (trace E). The concentration of the enzyme was 6 µM and the path length of the cell was 0.5 cm.
scattering. The high-frequency (1200-1700 cm-1) RR data contain several porphyrin modes termed as the oxidation state (ν4) and the ligation state (ν3, ν2, and ν10) modes. We have include the spectra of the oxidized (trace A) and reduced (trace B) enzyme for comparison with the spectrum of the CO-bound enzyme and those obtained from the NOR/NO and NOR/O2 reactions (see below).19 The spectrum of the CO-bound form of Nor (trace C) has prominent 6C/LS signals. Concurrent shifts exist for ν4 at 1373 cm-1, ν3 at 1494 cm-1, and ν10 at 1633 cm-1. It should be noted that ν2 coincides in frequency with other porphyrin modes such as ν11, ν37, and ν38, and thus, we are unable to assign it at present.19 When O2 is added to the CO-bound enzyme (trace D), the ν4 mode becomes a single band at 1373 cm-1 indicating oxidation of all hemes. With the exception of the strong bands at 1492 and 1572 cm-1, the spectrum of the reoxidized enzyme is very similar to the spectrum of the oxidized (as-isolated) enzyme. To determine if the heme b3-O-FeB bond is regenerated in the species we detect, we measured the RR spectrum (trace b) in the 700-900 cm-1 spectral region, shown in the inset. Trace a shows the spectrum of the as-isolated Nor in H2O. Comparison of traces a and b shows that the 813 and 833 cm-1 bands, that have been assigned to the open and closed forms of the heme b3-O-FeB dinuclear center, have not been regenerated. This observation indicates that the reoxidized enzyme is a 5C/HS His-heme b33+ species. Although the mechanism by which the enzyme activates O2 is not yet known, replacement of CO by O2 is evident. Following the initial binding of O2 to heme b3, the electron transfer from hemes b/c and the non-heme FeB to the heme b3 bound dioxygen
O2 and NO Sites of Cytochrome cbb3 and Nor
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Figure 2. High-frequency RR spectra of Nor in the oxidized (trace A), reduced (trace B), CO-bound (trace C), and the reaction product obtained by mixing oxygen with the CO-bound fully reduced Nor and allowing O2 to spontaneously replace CO (trace D). Inset: RR spectra of oxidized (trace a) and reoxidized (trace b) Nor. The excitation laser wavelength was 413.1 nm. The accumulation time was 10 min for each spectrum.
yields the 5C/HS His-heme b33+ species we have detected and H2O. Table 1 summarizes the frequencies of several RR modes of the Nor species shown in Figure 2. A close inspection of the RR data indicates that these frequencies are in agreement with earlier analyses of RR spectra of hemes b and c containing enzymes and with those of iron protoporphyrin model compounds.23,26 Trace A in Figure 3 was obtained by the direct addition of gaseous NO to the fully reduced Nor enzyme. Comparison of trace A with that of the fully reduced enzyme (trace B in Figure 2) shows significant differences. Trace A shows a split ν4 at 1362 and 1373 cm-1, three ν3 modes at 1484, 1492, and 1505 cm-1, two ν2 modes at 1565 and 1584 cm-1, and two ν10 modes at 1624 and 1639 cm-1. These modes indicate the presence of a mixture of heme b33+ and hemes b3+/2+ and c3+/2+. Trace B was obtained by subtracting a small portion of the fully reduced enzyme (trace B in Figure 2) from trace A to just avoid getting negative peaks. In the resulting spectrum, evidence for sixcoordination ferric heme b3 is self-evident. In particular, the bands at 1484 (ν3), 1565 (ν2), and 1624 cm-1 (ν10) indicate the presence of a ferric 6C/LS heme b3 species (see below). The bands at 1505 (ν3), 1584 (ν2), and 1639 cm-1 (ν10) demonstrate the presence of ferric 6C/LS hemes b/c species. Raman bands in the 200-800 cm-1 range include, in addition to vibrations of the porphyrin macrocycles, Fe-ligand motions
Figure 3. RR spectrum (trace A) of the fully reduced Nor/NO reaction. The spectrum was recorded immediately after the direct addition of gaseous NO to the fully reduced Nor enzyme. Trace B was obtained by subtracting a small portion of the fully reduced enzyme (trace B in Figure 2) from trace A to just avoid getting negative peaks. The excitation laser wavelength was 413.1 nm. The accumulation time was 10 min for each spectrum.
along the axis normal to the heme.19,25,27-29 Because the highfrequency RR data indicate the formation of new species, possibly nitrosylated heme b3 species, we have investigated the low-frequency spectral region. Figure 4 depicts the lowfrequency RR data of oxidized (trace A), reduced (trace B), and reaction products upon addition of 14NO (trace C) and 15NO (trace D) to the fully reduced enzyme. Addition of NO to the fully reduced enzyme causes small but noticeable changes in the RR spectra (traces C and D). Specifically, noticeable changes are observed in the 356, 393, and 445 cm-1 modes. The nitrosyl adducts of heme proteins in both the ferric and ferrous redox states exhibit the Fe-N-O vibrations in the 400-600 cm-1 region.19,25,30-35 The absence of a trough/peak in the difference spectrum (trace C - trace D), shown in the inset, clearly demonstrates that none of the changes we observe in the lowfrequency spectra are due to nitrosyl ligation to heme b3. Figure 5 shows the RR spectra of oxidized (trace A), reduced (trace B), and CO-bound (trace C) cbb3 oxidase and the spectrum (trace D) of the reaction product obtained by mixing oxygen with the CO-bound fully reduced cbb3 and allowing O2 to spontaneously replace CO. The spectrum from the oxidized enzyme has the ν4 mode at 1373 cm-1 indicating that all hemes are in the ferric state. The weak mode at 1473 cm-1 originates from ν3 and, in conjunction with ν2 at 1561 cm-1 and ν10 modes
TABLE 1: Frequencies of Several Resonance Raman Marker Modes (cm-1) in Nor oxidized
reduced
mode
5C/HS
6C/LS
5C/HS
6C/LS
ν2 ν3 ν4 ν10 ν11 ν37 νCdC
1578 1492 1373 1630
1584 1505 1373 1639
1560 1472 1362 1606
1584/1591 1494 1362
heme b3-CO 1584 1494 1373 1633
1550 1597 1624
1624
1624
reoxidized (O2) heme b3
reoxidized (NO) heme b3
5C/HS
6C/HS
1572 1492 1373 1630
1565 1484 1373
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Pinakoulaki and Varotsis TABLE 2: Frequencies of Several Resonance Raman Marker Modes (cm-1) in Cytochrome cbb3 Oxidase oxidized
reduced
heme b3-CO
mode 6C/HS 6C/LS 5C/HS 6C/LS ν2 ν3 ν4 ν10 ν11 ν37 νCdC
Figure 4. Low-frequency RR data of oxidized (trace A), reduced (trace B), and the reaction products upon addition of 14NO (trace C) and 15NO (trace D) to the fully reduced enzyme. Inset: trace C - trace D. The excitation laser wavelength was 413.1 nm. The accumulation time was 10-15 min for each spectrum.
1561 1473 1373 1628 1551
1584 1504 1373 1638
1555 1467 1362 1605
1592 1492 1362 1622 1545 1605
reoxidized (O2) heme b3 5C/HS
1592 1492 1373 1622
1572 1492 1373 1628
1622
density sensitive mode (ν4) of cbb3 in the fully reduced state (trace B) is at 1362 cm-1, establishing that all hemes are in the ferrous state. The well-isolated ν3 band at 1467 cm-1, ν2 at 1555, and ν10 at 1605 cm-1 establish the presence of a ferrous 5C/HS heme b3. The presence of ferrous 6C/LS hemes b and c is demonstrated by ν3 intensity at 1492 cm-1, by ν2 intensity at 1592 cm-1, and by ν10 intensity at 1622 cm-1. The band at 1605 cm-1 originates from ν37 of the low-spin hemes b and c. The ν11 mode is quite sensitive to axial ligation and geometric distortions of the heme macrocycle.23 The modes observed at 1545 and 1555 cm-1 are assigned to ν11 of hemes c and b, respectively. The near coincidence of the position of ν11 in heme b of cytochrome bo3 and heme b of the cytochrome cbb3 complex strongly suggests that they possess identical axial ligation (His-His) and heme geometries. Comparison of ν11 in heme c of cytochrome cbb3 with that of other cytochromes c, however, reveals significantly lower frequencies for heme c. This indicates that either the His-Met heme c configuration is perturbed from the rhombic symmetry observed in most cytochromes c or that the axial ligands in heme c of the cytochrome cbb3 complex are different from those found in other cytochromes c. Addition of CO to reduced cytochrome cbb3 causes a highspin to low-spin transition of heme b3. The formation of the heme b32+-CO complex is apparent from the shifts of ν4 from 1362 to 1373 cm-1 and of ν3 from 1467 to 1492 cm-1 (Figure 5, trace C). Addition of O2 to the CO-bound fully reduced ccb3 causes significant changes in the RR spectrum (trace D). The major changes all took place on a time scale shorter than 3 min. Therefore, the presence of ν3 at 1492 and ν2 at 1572 cm-1 demonstrates the rapid coordination of O2 to heme b3 and the subsequent heme b3 oxidation leading to a five-coordinate HisFe3+ heme b3. The presence of the ν3 band at 1504 cm-1, the ν2 band at 1584 cm-1, and the ν10 band at 1638 cm-1 demonstrates partial oxidation of the low-spin hemes c and b. The ν4 mode for reduced heme c at 1362 cm-1 indicates that not all of the hemes c are fully oxidized. The frequencies of these modes are summarized in Table 2. Discussion
Figure 5. RR spectra of oxidized (trace A), reduced (trace B), CObound (trace C) cytochrome cbb3 oxidase, and the spectrum (trace D) of the reaction product obtained by mixing oxygen with the CO-bound fully reduced cbb3 and allowing O2 to spontaneously replace CO. The excitation laser wavelength was 413.1 nm, and the concentration of the enzyme was 50 µM. The accumulation time was 5-10 min for each spectrum.
at 1628 cm-1, demonstrates that heme b3 is six-coordinate, highspin in its oxidized form. Also present in this spectrum are modes at 1504 (ν3), 1584 (ν2), and 1638 (ν10) cm-1 indicating the presence of 6C/LS hemes b and c. The porphyrin π* electron
The Nor/NO Reaction Products. Vibrational spectroscopy has been applied extensively to explore the Fe3+- and Fe2+NO complexes formed from the reactions of NO with heme and non-heme proteins, and thus, a large body of data is available.11,19,25,30-36 Understanding the interaction between NO and heme proteins is important for understanding the in vivo chemistry of NO and whether its reaction with heme Fe and non-heme Fe centers is dominated by a free radical character or by a behavior of Lewis base ligand. The end product of the Nor/NO reaction does not directly decay to the resting state since at 20 min after the reaction is initiated there is still evidence for a 6C/HS heme b3 species. We suggest that the
O2 and NO Sites of Cytochrome cbb3 and Nor end product decays to an oxidized form of the enzyme, which then undergoes a subsequent decay to the as-isolated form. It appears that there is a communication linkage, similar to that observed in the transition from the end product of the Nor/O2 reaction to the as-isolated form, between the distal and the proximal sites through bond networks. In both the Nor/O2 and Nor/NO reactions, H2O is released, and in the former case, the oxygen atom of the H2O molecule coordinates to the Fe of heme b3 to form the as-isolated enzyme whereas in the latter case the O atom coordinated to the non-heme FeB to form the as-isolated enzyme. In both cases, the structural change in the distal site upon H2O coordination is communicated to the proximal site through a polypeptide backbone leading to the rupturing of the His-Fe heme b3 bond. The molecular mechanism of NO reduction is not yet understood because it is difficult to obtain structural information on the state of the NO in the various intermediates that are formed during its reduction.37-38 In the past, most information has been obtained from optical and electron paramagnetic resonance (EPR) spectroscopies.14,16 In a model proposed by Moe¨nne-Loccoz and co-workers, the reaction is initiated when the dinuclear center is fully reduced and one molecule of NO binds to each metal center.17,18 They postulated that binding of NO to the ferrous heme b3 results in the dissociation of the proximal histidine. After dimerization, the bound NO molecules are reduced to N2O by using the available electrons on both metal centers. The reduction of 2NO to N2O leaves the ferric five-coordinate heme b3 bridged to the non-heme FeB. On the other hand, it has been proposed by Gro¨nberg et al.20 that the dinuclear site exists in a mixed valence form (heme b33+/nonheme FeB2+) prior to NO binding and that two NO molecules bind sequentially to the non-heme iron leaving the heme b3 essentially as a witness in the catalytic cycle. It has also been suggested that the bound NO acquires the character of nitroxyl anion NO-.16 This way, the dimerization of two such anions in the dinuclear center with subsequent protonation reactions and release of H2O would yield N2O. Furthermore, it was proposed that only nitroxyl anion can dissociate from the ferrous heme during catalysis, so that NO is effectively trapped by the heme after consumption of the reductant. As a result of this mechanism, the binuclear center is unlikely to become fully oxidized during the catalytic cycle in the absence of O2. The major issue in the reduction of NO to N2O is the formation of the N-N bond where diffusible, nontoxic intermediates capable of nitrosylation reactions are generated. We have recently proposed that if the physiological 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 FeB are reduced, and heme b3 is in the oxidized form, then addition of two electrons to Fe2+-NdO+ leads to the formation of Fe2+-Nd O- which subsequently upon nonenzymatic addition of NO is converted to N2O. The low potential of heme b3 (Em, pH 7.6 ) +60 mV) imposes a large thermodynamic barrier to reduction by the low-spin electron transferring heme b (Em, pH 7.6 ) +345 mV) and heme c (Em, pH 7.6 ) +310 mV)20 thus preventing the formation of a dead-end heme b32+-NO species as it was observed in the fully reduced cytochrome cbb3/NO reaction.25 On this line, we demonstrated that addition of NO to ferric heme b3 Fe yields Fe2+-NdO+.19 The experimental data presented here clearly demonstrate that the dead-end heme b32+-NO species is not populated, and the reduction of NO to N2O takes place when NO is coordinated to heme b3 in which the proximal histidine is ligated to the heme b3 Fe.
J. Phys. Chem. B, Vol. 112, No. 6, 2008 1855 It is instructive to compare our findings with the data available for cytochrome cbb3, which bears many similarities to Nor. The heme b32+-NO species in cbb3, with the NO and Fe-NO stretching modes at 1679 and 524 cm-1, respectively, was reported previously.25 These frequencies indicated that addition of NO to the fully reduced enzyme causes the cleavage of the His-Fe heme b3 bond producing a five-coordinate heme b3NO complex. The characterization of the ferrous nitrosyl species establishes the conditions under which the enzyme becomes locked into a dead-end species, because of the very slow dissociation of NO from this adduct. Under the same experimental conditions, the fully reduced Nor/NO reaction is not terminating in either a ferrous nitrosyl heme b32+-NO or a nitrosylated heme b3 species. Instead, the end product of the reaction is a 6C/HS species. The Cytochrome cbb3/O2 Reaction Products. A range of spectroscopic techniques has established that the initial dioxygen complex at the binuclear center of cytochrome c oxidase resembles the oxy complex of Mb and Hb.1,39-41 Most of the intermediate species formed in the cytochrome oxidase/O2 reaction have been well characterized.1,29,39-42 There is no consensus, however, on the intermediate and on the timing and mechanism of the critical O-O bond cleavage process in the heme a3/CuΒ binuclear center. A mechanism for the O-O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His-Tyr species to produce the oxoferryl species, CuΒ2+-OH-, and the Y280 radical has been proposed.43 We have shown recently, however, that the formation of the P intermediate, and thus the O-O bond cleavage process, can occur without the involvement of the cross-linked tyrosine.10 There is agreement, however, on the other intermediates during a single turnover reaction and that the hydroxy intermediate relaxes to pulsed and then to the resting state.39,42 Although the species we have detected here is the end product of multiple turnovers, the RR data indicate the formation of a high-spin oxidized form of heme b3. It is obvious, from our results, that there are some distinct differences between the oxidized form and the as-isolated (oxidized) enzyme. For instance, the ν2 mode in the oxidized form of the enzyme appears at ∼15 cm-1 higher than that of the as-isolated enzyme. This observation, in conjunction with the strong intensity of the ν3 vibration at 1492 cm-1, indicates that the species we detect undergoes further decay to the as-isolated form. We interpret this decay to a conformational change of the high-spin oxidized heme b3. Similar conclusions have been drawn for heme a3 in the cytochrome aa3/O2 reaction.39 It was demonstrated that the highspin form of the enzyme (pulsed) decays to a different highspin form (as-isolated) as a result of a conformational change of the high-spin heme a3. The presence of ferrous cytochrome c in the end product is not surprising since cytochrome cbb3 contains a total of six redox centers that could be involved in the four-electron reduction of O2 to H2O. As a result, the cbb3catalyzed reduction of molecular oxygen to H2O is completed without all the electrons available on the metal centers of the fully reduced enzyme. The Nor/O2 Reaction Products. The vibrational properties of the reaction product of the Nor/O2 reaction are best interpreted as indicating the formation of a 5C/HS species in which the proximal to heme b3 histidine ligand is intact. This way, the initial O2 association in the dinuclear heme b3-FeB center process involves binding and reduction to capture the substrate in the dinuclear center as a six-coordinate heme b3 hydroxy-species. Following this process and the conformational relaxation associated with the dinuclear center, the hydroxy intermediate
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exchanges rapidly with water, as it has been proposed in the aa3/O2 reaction, and its subsequent release from heme b3 produces the 5C/HS species we detect. Thus, the formation of the heme b3-O-FeB bond causes the rupture of the proximal heme b3-histidine bond and the reformation of the open and closed conformation of the oxidized 5C/HS heme b3-O(H)FeB dinuclear center. This is consistent with turnover experiments in which the enzyme was forced to turnover repeatedly, until the molecular O2 was depleted, terminating in a 5C/HS heme b3 Fe-O-FeB oxidized species.17
occurs without the proximal histidine, (2) the dinuclear center is unlikely to become fully oxidized during the catalytic cycle, and (3) the two NO molecules bind sequentially to the nonheme iron leaving the heme b3 essentially uninvolved are not consistent with our findings.
Conclusions
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The end products of the cbb3/O2 and Nor/O2 reactions reported here demonstrate the structural identity of the heme b3 in both enzymes prepared by the direct displacement of CO by O2. Thus, the presence of the non-heme FeB in Nor does not affect these late events in the O2 reduction pathway, although we cannot exclude the possibility that the kinetic properties including the decay of the primary Fe-O2 intermediate and the oxidation of the low-spin hemes c and b and the non-heme FeB are distinct in Nor. Considering the differences in the oxidized forms (as-isolated) of the enzymes, there must be structural differences in the heme b3-CuB and the heme b3-FeB centers that will only become evident in the properties of the transition from pulsed to oxidized, which must be clarified by further investigation. In the mechanism describing the structural implications involved in the transition from oxidized to reduced Nor and from oxidized to the NO-bound form and its photoproduct, it was demonstrated that there is a communication linkage between the distal and proximal sites through bond networks.19 It appears that similar conformational changes are involved in the transition from the pulsed enzyme to the oxidized enzyme (as-isolated). Finally, we conclude that the formation of the heme Fe-O-FeB bond is associated with the rupturing of the proximal to heme b3-His bond. In most O2-metabolizing enzymes, the electron transfer is ratelimiting, protonation is fast, and potentially toxic intermediates do not accumulate to significant level.1 In cytochrome oxidase, however, the proton transfers limit the reaction, the electrontransfer chemistry progressively slows down as the reaction proceeds, and tight coupling between the reduction process and proton translocation is assumed.1,29,40 As a consequence, partially reduced dioxygen intermediates rise transiently to levels that are detectable by spectroscopic techniques. Although Nor is not a proton pump, it is clear that the enzyme is capable of reducing O2 to H2O by utilizing the four redox metals and, thus, the reduction of O2 is completed with the electrons available on the metal centers.15 Direct examination of the reaction product of the Nor/NO reaction presented here leads to the conclusion that heme b3 is six-coordinate. Large changes in the RR of heme b3 would be expected if the iron-histidine bond were ruptured as a result of NO binding to heme b3. Similarly, large changes in the optical spectrum of heme b3 would be expected if the iron-histidine bond were ruptured, since in other heme b containing proteins it is established that the Soret maximum shifts from ∼420 nm to ∼390 nm upon going from six to five coordination when the nitrogeneous fifth ligand is lost. Taken together, we conclude that in the NO reduction process, the formation of the N-N bond occurs with the coordination of the proximal histidine to heme b3. Furthermore, the combination of both the optical absorption and RR data on the product of the Nor/NO reaction is fully consistent with the presence of an oxidized heme b3. Thus, the proposals that (1) the formation of the N-N bond
Acknowledgment. This work was partially supported by the Greek Ministry of Education and funds from the University of Cyprus. References and Notes
O2 and NO Sites of Cytochrome cbb3 and Nor (40) Varotsis, C.; Zhang, Y.; Appelman, E. H.; Babcock, G. T. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 237. (41) Ogura, T.; Takahashi, S.; Hirota, S.; Shinzawa-Itoh, K.; Yoshikawa, S.; Appelman, E. H.; Kitagawa, T. J. Am. Chem. Soc. 1993, 115, 8527.
J. Phys. Chem. B, Vol. 112, No. 6, 2008 1857 (42) Han, S.; Takahashi, S.; Rousseau, D. L. J. Biol. Chem. 2000, 275, 1910. (43) Proshlyakov, D. A.; Pressler, M.; Babcock, G. T. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8020.
