12860
J. Phys. Chem. B 2002, 106, 12860-12862
Fourier Transform Infrared Evidence for a Ferric Six-Coordinate Nitrosylheme b3 Complex of Cytochrome cbb3 Oxidase from Pseudomonas Stutzeri at Ambient Temperature Stavros Stavrakis, Eftychia Pinakoulaki, Andrea Urbani,† and Constantinos Varotsis* UniVersity of Crete, Department of Chemistry, 71409 Heraklion, Crete, Greece ReceiVed: August 15, 2002; In Final Form: NoVember 5, 2002
We report the first vibrational study of NO bound to an oxidized heme-copper oxidase. Cytochrome cbb3 oxidase from P. stutzeri reduces both O2 and NO to H2O and N2O, respectively. The ferric nitrosyl complex of cbb3 exhibits ν(N-O) at 1903 cm-1. This frequency is very similar to ν(NO) of nitric oxide reductase, the acidic form of Met Mb-NO, but 18 cm-1 lower than that of neutral Met Mb-NO. By monitoring the NO intensity, we estimate that NO dissociates from the heme b3 pocket, without binding to CuB, with k ) 1.8 × 10-3 s-1. Therefore, NO binding occurs at the heme site and not at CuB, generating a nitrosonium CuB1+NO+ species as proposed recently (Torres, J.; Cooper, C. E.; Wilson, M. T. J. Biol. Chem. 1998, 273, 87568766). The coordination of NO to cbb3 oxidase and to nitric oxide reductase and Mb is compared and discussed.
Introduction Nitric oxide (NO) plays a diverse role in regulating a range of physiological processes in higher organisms such as the immune response to tumor cells, neuronal synaptic transmission, and cytochrome c oxidase activity.1,2 Recently, it was demonstrated that the NO reductase activity of cbb3 oxidase (Km(NO) ) 12 µM), although lower than that measured for its structurally related nitric oxide reductase (Nor), is the highest among the heme-copper oxidases.3 It has been proposed that in bacterial NO reductases and in cytochrome cbb3 oxidase the mechanisms of O2 and NO reduction share common features and that NO and O2 may used as alternative substrates by both enzyme families.4 The mechanism by which cbb3 oxidase selectively reduces O2 to H2O and NO to N2O provides the key to understanding the possible co-evolution of aerobic respiration and denitrification. Cytochrome cbb3 oxidase contains three low-spin hemes c and one low-spin heme b that are involved in the electron pathway to the binuclear heme b3/CuB center where O2 and NO are reduced to H2O and N2O, respectively.3,5 The binuclear center in cbb3 is unique when compared to those of aa3-type oxidases; it lacks the hydroxyethylfarnesyl side chain and the highly conserved among the heme-copper oxidases tyrosine 244, both of which have been proposed to play a crucial role in the properties of the binuclear center.6 The resonance Raman studies of the fully reduced5 and CO-bound6 forms of cbb3 oxidase have revealed several structural characteristics of the heme pocket, including an indication from the frequencies of Fe-CO and Fe-C-O that the binuclear center has a unique active site with a more open pocket than that in other hemecopper oxidases. Recently,7 we demonstrated by time-resolved step-scan FTIR experiments that in cbb3-type oxidases the decay of the transient CuB-CO complex, in contrast to the aa3-type, is concurrent with the formation of the heme b3-CO complex and that ν(CO) of CuB at 2065 cm-1, despite the lack of the * Corresponding author. E-mail:
[email protected]. Fax: 32-810393601. † Present address: Clinical Biochemistry Laboratory, Ospedale del Bambino Gesu' IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
cross-link tyrosine 244, is similar to that observed in cytochrome aa38-10 and cytochrome bo3.11 The reaction of NO with the oxidized heme-copper aa3-type oxidase (CcO) has been investigated by optical spectroscopy.2 It was suggested that NO reacts with CuB2+, generating a nitrosonium species CuB1+NO+ that upon hydration yields nitrous acid and CuB1+ and thus nitrosylation at CuB1+ instead of at heme a3 is the key event in the fast inhibition of CcO by NO. Moreover, several mechanisms were proposed for the reaction of NO with the oxygen intermediates P and F, and the results were rationalized through a mechanism in which NO reacts with CuB2+. The first step in understanding the cytochrome cbb3/NO reaction is the characterization of the binuclear center upon NO binding. In this work, we extend our previous investigation7,12 and report the NO-bound form of oxidized cbb3 by Fourier transform infrared spectroscopy (FTIR) and compare it with that of the heme b33+-NO complex of Nor and the NO complex of metmyoglobin at pH 4 and 7.5. Consideration of the data on ferric and ferrous nitrosyl adducts of other heme proteins indicates that this unstable species is the heme b33+-NO complex. This observation contrasts sharply with the conclusions drawn for the aa3-type oxidases and the proposed mechanisms for the reaction of NO with oxygen intermediates in hemecopper oxidases. By monitoring the NO intensity, we estimate that NO dissociates from the heme b3 pocket with k ) 1.8 × 10-3 s-1. The coordination of NO to cbb3 oxidase and to Nor and Mb is compared and discussed. Materials and Methods Cytochrome cbb3 oxidase from P. stutzeri3 and Nor from P. denitrificans13 were purified as described elsewhere. Mb was purchased from Sigma, and NO gas was obtained from Messer (Frankfurt, Germany). FTIR spectra were obtained from 200 to 300 µM samples with a Bruker Equinox 55 FTIR spectrometer equipped with a liquid-nitrogen-cooled MCT detector. The samples were loaded anaerobically into a cell with CaF2 windows and a 0.025-mm spacer. Difference spectra were obtained by using the buffer as a background, and each spectrum is the average of 200 scans. The spectral resolution used for the FTIR measurements was 4 cm-1 for all spectra. Optical
10.1021/jp026763l CCC: $22.00 © 2002 American Chemical Society Published on Web 11/21/2002
Letters
J. Phys. Chem. B, Vol. 106, No. 50, 2002 12861 TABLE 1: Frequencies (cm-1) of the N-O Stretching Frequencies in Heme Protein-NO Adductsa molecules
ν(NO)
ref
Mb (pH 7) Mb (pH 4) Mb (pH 4) HbA HRP NP Nor cd1 cbb3 (pH 5.5) cbb3 (pH 7.5)
1922 1910 1907 1925 1903 1904 1904 1910 1903 1903
15 and 16 15 this work 19 15 18 14 17 this work this work
a Abbreviations: Mb ) myoglobin; HbA ) hemoglobin A; HRP ) horseradish peroxidase; NP ) nitrophorin; Nor ) nitric oxide reductase; cd1 ) cytochrome cd1; cbb3 ) cytochrome cbb3.
Figure 1. (A) Optical difference spectra of NO-bound cytochrome cbb3 minus oxidized cbb3 from Pseudomonas stutzeri 5, 10, and 15 min after addition of NO. (B) (I) Time evolution of the FTIR spectra of the NO-bound cytochrome cbb3 at pH 7.5; (II) Kinetic analysis of the 1903-cm-1 mode.
Figure 2. FTIR spectra of cytochrome cbb3-NO at pH 5.5 (A) and 7.5 (B), nitric oxide reductase-NO (C), and Met Mb-NO at pH 7.5 (D) and 4 (E). Met Mb-NO at pH 4 was prepared by the addition of acetic acid into the pH 7 Met Mb-NO solution.
absorbance spectra were recorded before and after the FTIR measurements in order to assess sample stability with a PerkinElmer Lamda 20 UV-visible spectrometer. Results and Discussion The optical difference NO-bound cbb3-oxidized spectra shown in Figure 1A display a positive band at 419 nm and a trough at 406 nm. At 15 min after NO addition, the peak-totrough intensity has diminished, indicating the dissociation of the heme b33+-NO species. The FTIR spectra of the NO-bound oxidized cbb3 at pH 7.5 displayed in Figure 1B (I) show a single vibration at 1903 cm-1, and its frequency remains unchanged at pH 5.5 (Figure 2, trace A). This frequency is very similar to ν(NO) of Nor14 (Figure 2, trace C), the acidic form of Met MbNO15 (Figure 2, trace E), but is 18 cm-1 lower than that of the neutral Met Mb-NO (Figure 2, trace D).15,16 In addition, it is very similar to those of heme cd117 (1910 cm-1) and nitrophorin18 (1904 cm-1). Table 1 tabulates the N-O stretching frequencies of several heme proteins. Accordingly, we assign the 1904 cm-1 mode to ν(NO) of the ferric NO adduct of cbb3.
The time evolution of the NO mode shown in Figure 1B (I) indicates, in agreement with the optical data, that NO dissociates from the heme pocket with k ) 1.8 × 10-3 s-1 (Figure 1B (II)). ν(NO) of Met Mb-NO is pH-dependent. As suggested by Miller et al.16 and Tomita et al.,15 the stability of the Fe-NO structure depends on the protonation of the distal His. At low pH, the distal His swings out into the solvent, stabilizing the Fe+dNdO structure (νNO ) 1907 cm-1). If the lone pair of the distal His is located close to the O atom of the bound NO, then the Fe-NtO+ structure is stabilized, and ν(NO) is raised (νNO ) 1921 cm-1). It was also demonstrated by Tomita et al.15 for four heme proteins containing histidine as a proximal ligand that the NO moiety is electron deficient and that the effect of π donation from the proximal His is small. ν(NO) of cbb3 is pH-independent, indicating that the closest residues with labile protons to heme b3, which are the His ligands of CuB, have no direct effect on the properties of the heme b33+-NO complex. The ν(NO) at 1903 cm-1 that we observe is consistent with a linear heme b3 Fe-N-O unit in which a substantial donation of charge from NO to heme b3 Fe3+ has occurred, resulting in a species with considerable NO+ character and an elevated ν(NO) stretching frequency as compared to those of free NO (1875 cm-1) and ferrous NO (1590-1681 cm-1). Linear Fe2+NtO+ character means that partial charge transfer from NO to Fe3+ occurs during the bonding process. Furthermore, the optical data indicate that the heme b32+-NtO+ complex, although NO is a 3e- donor and in conjunction with a H+ can form the proper Fe-N-O orientation to push electrons facilitating the Fe reduction, is not converted to a ferrous heme b32+-NdO species but rather decays to the oxidized form of the enzyme. Similar observations have been reported for the nitrosyl complexes of nitrophorins (NP1-NP3)20 and Mb.21 Other ferric heme proteins such as Met Mb21 and nitrophorins20 can bind and release NO. The binding of NO to heme b3 of Nor, however, is essentially irreversible.14 The difference in the kinetics between nitrosyl-cbb3 and Nor is not related to their common function since both enzymes catalyze the reduction of NO to N2O. A low midpoint redox potential has been measured for heme b3 of cbb3, indicating that full reduction of the binuclear center is thermodynamically unfavorable.22 Thus, it is possible that under physiological conditions NO activation in cbb3 occurs with a mixed-valence form of the enzyme in which the low-spin hemes b and c are reduced and heme b3 is in its oxidized form. This way, NO binds to heme b3, and the addition of two electrons to heme b3 Fe+-NO prior to its dissociation yields the two-electron reduced species Fe2+-Nd O-. A second NO molecule attacks the N atom of the ferrous NO species to yield hyponitrite (HONNO-) transiently, thus
12862 J. Phys. Chem. B, Vol. 106, No. 50, 2002 the N-N bond formation. Cleavage of the N-O bond produces the ferric enzyme, N2O, and H2O. The data reported here and those reported earlier on the fully reduced cbb3/NO reaction12 demonstrate that NO binding occurs exclusively at the heme b3 center and not at CuB,2 as previously reported. This is the first report of the vibrational spectrum of NO bound to an oxidized heme-copper oxidase. We postulate that a more open pocket in cbb3 than in Nor lowers the energy barrier, thus optimizing the efficiency of NO escape. If the differences in the kinetics of NO-bound heme b3 of cbb3 and Nor are a reflection of the properties of the distal environment of heme b3, then association and dissociation reactions with NO under different chemical conditions (pH, D2O) coupled with a structural analysis such as that reported here will allow for a quantitative determination of the importance of distal and proximal effects on the ligand-binding and ligand-release processes. Although we report the formation of an unstable nitrosyl heme b3 species that can be a real intermediate in the catalytic pathway, the mechanism of NO reduction to N2O by cbb3 appears to be complex. To fully understand the catalytic mechanism of heme-copper oxidases, all of the various possible intermediate states must be characterized. Additional studies of NO coordination to mixed-valence cbb3 are necessary to formulate a complete mechanism for the reduction of NO to N2O under physiological conditions. Experiments on this point are in progress. Acknowledgment. This work was supported in part by the Greek Ministry of Education. References and Notes (1) Bredt, D. S.; Snyder, S. H. Annu. ReV. Biochem. 1994, 23, 175195. (2) Torres, J.; Cooper, C. E.; Wilson, M. T. J. Biol. Chem. 1998, 273, 8756-8766.
Letters (3) Forte, E.; Urbani, A.; Saraste, M.; Sarti, P.; Brunori, M.; Giuffre, A. Eur. J. Biochem. 2001, 268, 1-6. (4) Giuffre`, A.; Stubauer, G.; Sart, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14718-14723. (5) Varotsis, C.; Babcock, G. T.; Garcia-Horsman, A.; Gennis, R. B. J. Phys. Chem. 1995, 99, 16817-16820. (6) Wang, J.; Gray, K. A.; Daldal, F.; Rousseau, D. L. J. Am. Chem. Soc. 1995, 117, 9363-9364. (7) Stavrakis, S.; Koutsoupakis, K.; Pinakoulaki, E.; Urbani, A.; Saraste, M.; Varotsis, C. J. Am. Chem. Soc. 2002, 124, 3814-3815. (8) Dyer, R. B.; Einarsdo´tirr, O.; Killough, P. M.; Lopez-Garriga, J. J.; Woodruff, W. H. J. Am. Chem. Soc. 1989, 111, 7657-7659. (9) Park, S.; Pan, L.-P.; Chan, S. I.; Alben, J. Biophys. J. 1996, 71, 1036-1047. (10) Iwase, T.; Varotsis, C.; Shinzawa-Itoh, K.; Yoshikawa, S.; Kitagawa, T. J. Am. Chem. Soc. 1999, 121, 1415-1416. (11) Puustinen, A.; Bailey, J. A.; Dyer, R. B.; Mecklenberg, S. L.; Wikstrom, M.; Woodruff, W. H. Biochemistry 1997, 36, 13195-13200. (12) Pinakoulaki, E.; Stavrakis, S.; Urbani, A.; Varotsis, C. J. Am. Chem. Soc. 2002, 124, 9378-9379. (13) Hendriks, J.; Warne, A.; Gohlke, U.; Haltia, T.; Ludovici, C.; Lu¨bben, M.; Saraste, M. Biochemistry 1998, 37, 13102-13109. (14) Pinakoulaki, E.; Gemeinhardt, S.; Saraste, M.; Varotsis C. J. Biol. Chem. 2002, 277, 23407-23413. (15) Tomita, T.; Haruta, N.; Aki, M.; Kitagawa, T.; Ikeda-Saito, M. J. Am. Chem. Soc. 2001, 123, 2666-2667. (16) Miller, L. M.; Pedraza, A. J.; Chance, M. R. Biochemistry 1997, 36, 12199-12207. (17) Wang, Y.; Averill, B. J. Am. Chem. Soc. 1996, 118, 3972-3973. (18) Ding, X. D.; Weichsel, A.; Andersen, J. F.; Shokhireva, T. K.; Balfour, C.; Pierik, A. J.; Averill, B. A.; Montfort, W. R.; Walker, F. A. J. Am. Chem. Soc. 1999, 121, 128-138. (19) Sampath, V.; Zhao, X. J.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1994, 198, 281-287. (20) Ribeiro, J. M. C.; Hazzard, J. M. H.; Nussenzveig, R. H.; Champagne, D. E.; Walker, F. A. Science (Washington, D.C.) 1993, 260, 539-541. (21) Leverman, L. E.; Wanat, A.; Oszajca, J.; Stochel, G.; Ford, P. C.; van Eldik, R. J. Am. Chem. Soc. 2001, 123, 285-293. (22) Pereira, M. M.; Carita, J. N.; Anglin, R.; Saraste, M.; Texeira, M. J. Bioenerg. Biomembr. 2000, 32, 143-152.
