Resonance Raman and FTIR Studies of Carbon Monoxide-Bound

Eftychia Pinakoulaki , Constantinos Koutsoupakis , Hitomi Sawai , Andrea ... Vangelis Daskalakis, Eftychia Pinakoulaki, Stavros Stavrakis, and Constan...
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7670

J. Phys. Chem. B 1998, 102, 7670-7673

Resonance Raman and FTIR Studies of Carbon Monoxide-Bound Cytochrome aa3-600 Oxidase of Bacillus subtilis Constantinos Varotsis* and Magdalini Vamvouka UniVersity of Crete, Department of Chemistry, 71409 Iraklion, Crete, Greece ReceiVed: May 28, 1998

Resonance Raman and FTIR spectra are reported for the fully reduced carbon monoxy derivative of the quinol aa3-600 oxidase from Bacillus subtilis. The resonance Raman spectra display two isotope-dependent vibrational modes at 520 and 575 cm-1. The FTIR spectrum displays a single vibrational mode at 1963 cm-1. We assign the band at 520 cm-1 to the Fe-CO stretching mode, the band at 575 cm-1 to the FeC-O bending mode, and the band at 1963 cm-1 to the C-O stretching mode. The frequencies of these modes are similar to those that have been reported for the CO-bound mammalian cytochrome c oxidase. Despite the fact that two different heme-protein conformations that affect the iron-his bond strength are present in the ferrous ligand-free form of aa3-600, the CO-bound adduct has a single conformation in which the His-Fe-CO CuB moiety has the same structure as the R form found in the mammalian cytochrome c oxidase. The present and previous data on the vibrational frequencies of ferrous ligand-free and ferrous CO-bound forms of terminal oxidases show that an inverse linear relationship exists between the frequencies of the Fe-his and Fe-CO stretching modes. We suggest that the frequencies of both the Fe-CO and C-O modes found in heme-CuB oxidases are affected by the proximal His376, which is H-bonded to the peptide carbonyl of Gly351, and by distal effects on the heme a3-bound CO exerted by CuB.

Cytochrome aa3-600, isolated from the Gram-positive bacterium Bacillus subtilis, is a member of the superfamily of structurally related respiratory heme-copper oxidases.1-3 It is a hydroquinone oxidase and, recently, was shown to function as a proton pump, as well as to catalyze the reduction of dioxygen to water through intermediates similar to those that occur in cytochrome c oxidase.4,5 The aa3-600 oxidase, which is grown on a fermentable carbon source and with moderate aeration, is unique in several respects. First, unlike the other proteins in the terminal oxidase superfamily, such as mammalian cytochrome c oxidase which possesses four metal centers, this quinol oxidase contains only three catalytically active centers: heme a, which comprises the electron delivery site, and heme a3-CuB designated as the site of oxygen reduction and ligand binding.1,3 In contrast to the mammalian enzyme which has been shown to display a single Fe-his stretching mode, the fully reduced unliganded form of aa3-600 is characterized by two distinct iron-histidine stretching modes which have been proposed to arise from two different heme-protein conformations that affect the iron-histidine bond strength.6 In addition, the reduced enzyme has an exceptional R-band absorption maximum that is ∼7 nm blue shifted relative to most other oxidases of the aa3-type.1,4 Structural information on the heme-CuB center in the quinol and cytochrome c oxidase superfamily has been determined from studies of their CO-bound adducts. In addition to revealing insights concerning the geometry of the bound CO, the identity of the proximal ligand, as well as the interactions between the bound CO and the surrounding residues in the heme pocket, CO photodissociation and recombination studies have been very powerful for studying the conformational and structural changes as well as the kinetic properties of transient species.14a,b,15 The * Corresponding author. Fax: 30-81-210951. E-mail: [email protected].

vibrational frequencies of the FeCO unit obtained by resonance Raman (RR) and FTIR spectroscopies, which include the FeCO stretching mode (520 cm-1), the Fe-C-O bending mode (575 cm-1), and the C-O stretching mode (1962 cm-1) have been identified in aa3- and bo3-type oxidases.7,9,16-18 (See Table 1.) Photodissociation studies have shown that the Fe-his mode is strengthened on a time scale of up to ∼1 µs subsequent to CO photolysis and that CO recombination occurs at ∼1 ms. Despite the presence of a proximal histidine in both the a3- and o3-type oxidases, the frequencies of ν(Fe-CO) and ν(C-O) deviate significantly from the inverse linear relationship curve that exists between the frequencies of these two modes in histidine-coordinated heme proteins.11 The origin for the displacement of the carbon monoxide complexes of heme/Cu terminal oxidases has not yet been determined, although it was postulated to result from distal effects that arise, presumably, from CuB and its ligand set13 or from a strained proximal histidine-Fe-CO complex.7 FTIR studies of the ν(C-O) mode of mammalian aa3-type cytochrome c oxidase at 10 K revealed two major conformations, termed R- and β-forms, at the active site.17 In the R form described above, the ν(C-O) mode was detected at 1964 cm-1, whereas for the β-form the corresponding mode was located at 1952 cm-1. More recently, observation of both the R- and β-forms in the aa3-type oxidase of R. sphaeroides at room temperature has been reported.11 On the other hand, in cbb3type oxidase only the β-form was observed.8 Unlike the R-form, which deviates significantly from the linear correlation curve, the frequencies of the β-form of the aa3-type oxidase of R. sphaeroides place it on the ν(Fe-CO) versus ν(C-O) curve. Unfortunately, the functional significance of the R- and β-forms of the terminal oxidases has not been determined yet. Resonance Raman (RR) scattering has been applied to the study of terminal oxidases and has been found to be a powerful

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Carbon Monoxide-Bound Cytochrome aa3-600 Oxidase

J. Phys. Chem. B, Vol. 102, No. 39, 1998 7671

TABLE 1: Fe(II)-His, Fe-CO, Fe-C-O, and C-O Frequencies (cm-1) for Some Heme-Copper Respiratory Oxidasesa complexes cytochrome aa3-600 cytochrome c oxidase (aa3) cytochrome ba3 cytochrome bo3 cytochrome cbb3 a

ν(Fe(II)-His) ν(Fe-CO) δ(Fe-C-O) ν(C-O) 194/2146 21419,20 193/20923 20829 23528

520a 5207 1974/198322 5249 4958

575a 5787

1963a 196318

5779 5748

196037 195038

This work.

technique in probing the bonds of the hemes and the bonds between the iron atom and the proximal or the axial ligands.19,20 RR studies of aa3-600 revealed strong similarities of the ligation and heme environments with those found in the mammalian enzyme.6 However, the low-frequency region of the RR spectrum showed two modes at 194 and 214 cm-1 in the fully reduced aa3-600, while a single vibration at 214 cm-1 has been assigned to the iron-histidine stretch of heme a3 in mammalian cytochrome c oxidase that is known from crystallographic studies to be H-bonded to Gly351.21 The occurrence of two ν(Fe-his) frequencies, with one being unusually low, indicates unique structural features associated with the proximal ligation of heme a32+ in the aa3-600 which could be critical to function. Therefore, studies of this bacterial enzyme increase our understanding of the structural and functional properties of all terminal oxidases. In the work presented here, the carbon monoxy derivative of the aa3-600 quinol oxidase has been characterized by RR and FTIR spectroscopies. Our results indicate that, despite the presence of two proximal iron-histidine modes in the ferrous ligand-free form of the enzyme, in the heme a3-bound CO complex the frequencies of the modes involving CO do not split, as was observed in cytochrome ba3,22,23 but rather are identified as ν(Fe-CO), δ(Fe-C-O), and ν(C-O) at 520, 575, and 1963 cm-1, respectively. This shows that the his-Fe-CO CuB moiety is similar to the R-form found in other aa3-type terminal oxidases. Moreover, we have used the frequencies of the FeCO and Fe-his modes of several terminal oxidases to demonstrate that an inverse linear correlation exists between the frequencies of these two modes. We suggest that both the proximal His376, which is H-bonded to Gly351, and the effects exerted by CuB on the heme a3-bound CO are the key determinants of the frequencies observed for the Fe-CO and C-O stretching modes in heme-CuB oxidases. Materials and Methods Cytochrome aa3-600 was isolated as described by Lauraeus et al.1 The fully reduced carbonmonoxy samples were prepared by adding a few grains of sodium dithionite, 15 mM ascorbate, and 1 mM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) to the oxidized enzyme and then flushed with CO gas. Resonance Raman spectra were obtained from 30-40 µM samples, pH 7.5, in a cylindrical quartz spinning cell maintained at 3-5 °C by a stream of cold nitrogen gas. The Raman spectra were acquired by using a SPEX 1877 triplemate with an EG&G (model 1530-CUV-1024S) CCD detector. A Coherent Innova K-90 Krypton ion laser was used to provide the excitation wavelength of 413.1 nm. The power incident on the oxidase samples was typically 2-4 mW. FTIR spectra were recorded from 180-250 µM samples at 2 cm-1 resolution with a BRUKER Equinox 55 FTIR spectrometer equipped with a liquid nitrogen cooled mercury cadmium telluride detector. The carbonmonoxy samples were loaded into a cell with CaF2

Figure 1. Resonance Raman and FTIR spectra (inset) of the CO-bound form of quinol aa3-600. The excitation laser wavelength was 413.1 nm. The 12C16O-bound and the 13C16O-bound forms are shown in A and B, respectively.

windows and a 0.052-mm spacer. An average of 2100 scans was used for each spectrum. Optical absorption spectra were recorded before and after FTIR and Raman measurements in order to assess sample stability with a Perkin-Elmer Lamda 20 UV-vis spectrophotometer. Results and Discussion Figure 1 shows the low- to mid-frequency RR spectra and the FTIR spectrum (inset) of the carbon monoxy complexes of cytochrome aa3-600. We detect two bands in the RR spectrum, one at 520 cm-1 and the other at 575 cm-1, which display carbon isotopic sensitivity by shifting to 514 and 559 cm-1, respectively, in the 13CO-bound adduct. We assign the band at 520 cm-1 to the Fe-CO stretching mode and the band at 575 cm-1 to the Fe-C-O bending mode. We also assign the FTIR band located at 1963 cm-1 (∆ν1/2 ) 8.1 cm-1), shown in the inset in Figure 1, to the C-O stretching mode in the 12C16O-bound form of the fully reduced enzyme. The frequencies of these FeC-O modes are very close to those that have been reported for the aa3-type cytochrome c oxidase from beef heart7,12 and Rhodobacter sphaeroides,11,13 as well as quinol cytochrome bo3 from Escherichia coli,9 with the exception of the Fe-C-O bending mode which is 4 cm-1 lower in cytochrome aa3-600 than it is in cytochrome aa3. The ratio of the intensity of the Fe-C-O bending mode to that of the Fe-CO stretching mode, however, is close to that found in cytochrome aa3 (Iδ/Iν ) 0.20.4). RR studies of heme model compounds have revealed that the intensity of the bending vibration is proportional to the sterically induced tilting of the CO away from the heme plane.24 Therefore, the Fe-C-O moiety in the aa3-600 is similar to the bovine aa3-CO complex in that the CO is tilted by 21° with respect to the heme plane.25 The present RR and FTIR results provide clear evidence that CO binds in a single conformation to heme a3, even though two conformers have been detected in the ferrous ligand-free form of the enzyme. If the CO ligand was bound in two distinct conformations, a second pair of ν(Fe-CO) and ν(CO) modes should appear in the RR and FTIR spectra. They were, however, not observed. The single conformer detected is essentially the R-form, exhibiting ν(Fe-CO), δ(Fe-C-O), and ν(C-O) modes at 520, 575, and 1963 cm-1, respectively, spectral behavior that is similar to that observed for mammalian oxidase10,11 in which His376, the proximal ligand of heme a3,

7672 J. Phys. Chem. B, Vol. 102, No. 39, 1998

Figure 2. Correlation between frequencies of the Fe-CO versus the Fe-his stretching modes. (b) Mammalian cytochrome c oxidase7,18-20 and aa3-6006 (this work), (9) cytochrome bo3,9,29,37 and (2) cytochrome cbb3.8,28,38

is H-bonded to the peptide carbonyl of Gly351. This provides strong evidence that, in aa3-600, CO binding to heme a3 shifts the his-Fe-CO conformational equilibria toward the H-bonded proximal histidine conformer. On the other hand, split ironhistidine and CO stretching modes have been assigned in the RR and FTIR spectra of ferrous ligand-free and ferrous CObound forms of cytochrome ba3, indicating the presence of two conformers.22,23 This behavior can be reasonably interpreted as a consequence of proximal-side changes, as discussed below. It is well-known from RR studies that the properties of the trans ligand of carbonmonoxy-heme complexes can affect bonding between the iron and the distal CO and, thus, its vibrational frequency.26 CO coordinates to the heme iron through π-back-bonding from the iron dπ orbitals to the π* orbital of the CO and through a σ bond formed by the lone pair on the CO and the iron dz2 orbital. An increase in the dπ-π* overlap increases the Fe-CO bond order and concomitantly decreases the C-O bond order.27 However, the σ-bonding interactions of the proximal and CO ligands compete for the same Fe dz2 orbital. Therefore, stronger σ-bonding from the proximal Fe-N(his) is expected to weaken the Fe-CO bond. This argument is consistent with RR studies of carbonmonoxy model compounds, which indicate that strengthening of the proximal-axial ligand bond weakens the Fe-CO of the bound CO.26,27 Figure 2 shows that an inverse linear relationship exists between the frequencies of Fe-his and Fe-CO stretching modes in the terminal oxidases. The ν(Fe2+-his) of heme a3, heme o3, and heme b3, shown in Figure 2, fall between 208 and 235 cm-1.20,28,29 Such alteration in the strength of the Fe-his mode can be attributed to the strength of the H-bond of the proximal His376 ligand to Gly351. Therefore, the observed ∼20 cm-1 downshift in the ν(Fe-CO) of cytochrome cbb3,8 when compared to that found in cytochrome aa3,19,20 is brought about by an increase in the imidazolate character of the proximal histidine. This argument is supported by the observed high frequency of the Fe-N(his) stretching mode at 235 cm-1, as compared to the frequencies of the aa3-19,20 and bo3-type29 oxidases, which has been attributed to strong hydrogen bonding of the proximal histidine. Therefore, in cytochrome cbb3 the increased electron density at Fe increases back-bonding, as reflected in the lowered ν(C-O), but the increase in Fe-CO bond order is overriden

Varotsis and Vamvouka by competition between the strongly H-bonded histidine and the CO for the Fe σ orbital (dz2), leading to lowering of ν(FeCO). The structural interpretation outlined above specifies a hydrogen-bonding interaction of His376 with the carbonyl of Gly351 as the source for the strength of the Fe-CO and C-O stretching modes found in heme-CuB oxidases. Moreover, ab initio calculations have shown that the proximal histidine largely determines the CO distortion in carbonmonoxy globin proteins.30 On the other hand, RR studies of the CO-bound aa3-type cytochrome c oxidase from R. sphaeroides revealed a ν(FeCO) in the His333Asn mutant, which is a ligand to CuB, that was substantially lower than that observed for the CO complex of the wild type.13 It was proposed that distal effects, due to the proximity of CuB to the heme a3 bound CO, are likely to account for the high frequency of the Fe-CO stretching mode and the position of oxidases off the ν(Fe-CO) versus ν(C-O) correlation curve. Here, the situation contrasts somewhat with what has been observed for the oxy intermediate in the cytochrome/dioxygen reaction,31-33 as the ν(Fe-O2) in the oxyheme a32+ species is very similar to that observed for the oxy complexes of the O2 transport proteins.34,35 These observations indicate little perturbation of the bound ligand by heme a32+ pocket effects. It is noteworthy that neither the ν(FeCO) nor the ν(CO) have shown frequency shifts upon H2O/ D2O exchange.36 Our data in conjunction with those obtained by others13 suggest that in heme-CuB oxidases the strength of the proximal histidine H-bonding interaction affects the strength of both the Fe-C and C-O bonds which could be further influenced by the CuB distal environment. The molecular origin for the single FeCO conformer in the aa3-600 oxidase as compared to ba3 oxidase is intriguing. The existence of two conformers in ba3 oxidase was observed in the the ν(C-O) and ν(Fe-his ) regions of its FTIR and RR spectra, respectively.22,23 The relative intensities of the Fehis stretching modes were found to be temperature dependent with the same thermodynamic parameters as those of the C-O stretch. Thus the splitting of the Fe-N and C-O stretching modes was attributed to the same conformational effects. In the aa3-600, however, the binding of CO to heme a3 changes the conformation of the proximal residues in a manner that favors the hydrogen-bonded his-Fe-CO conformer. This conformational change can also act as a control mechanism for the coordination and redox chemistry of heme a3. In addition, the strength of the hydrogen bond would also regulate the anionic character of the proximal histidine affecting the stabilization of higher oxidation states of the heme a3 Fe(IV)d O that form as part of the catalytic pathway of the enzyme. The RR and FTIR results presented here establish that, despite the existence of two conformations in the fully reduced aa3600, the CO ligand binds to heme a3 in a single geometry, which is similar to that observed in mammalian aa3 oxidase, giving rise to one conformer (R-form). The data presented in this study and previously suggest that the ν(Fe-CO) and ν(C-O) frequencies in the terminal heme/Cu oxidases are affected from both the distal and proximal environments. The thermodynamics of the two conformers in the fully reduced form of the aa3600 and the mechanism by which the protein controls the chemistry of the binuclear heme a3/CuB site is under investigation. Acknowledgment. We are indebted to Prof. Gerald Babcock for the use of the LASER laboratory facility at Michigan State University and Prof. M. Wikstro¨m for supplying the aa3-600 samples. This work was supported by Alexander von Humboldt-

Carbon Monoxide-Bound Cytochrome aa3-600 Oxidase Stiftung, DFG (Grant SFB 472), Fonds der Chemischen Industrie, a NATO grant (Grant GRG 940275), and PENED95. References and Notes (1) Lauraeus, M.; Haltia, T.; Saraste, M.; Wikstro¨m, M. Eur. J. Biochem. 1991, 197, 699-705. (2) Powers, L.; Lauraeus, M.; Reddy, K. S.; Chance, B.; Wikstro¨m, M. Biochim. Biophys. Acta 1994, 113, 504-512. (3) Lauraeus, M.; Wikstro¨m, M. J. Biol. Chem. 1993, 268, 1147011473. (4) Lauraeus, M.; Morgan, J. E.; Wikstro¨m, M. Biochemistry 1993, 32, 2664-2670. (5) Varotsis, C.; Lauraeus, M.; Babcock, G. T.; Wikstro¨m, M. Biochim. Biophys. Acta 1995, 1231, 111-116. (6) Lauraeus, M.; Wikstro¨m, M.; Varotsis, C.; Tecklenberg, M. M. J.; Babcock, G. T. Biochemistry 1992, 31, 10054-10060. (7) Argade, P. V.; Ching, Y.-c.; Rousseau, D. L. Science 1984, 225, 329-331. (8) Wang, J.; Gray, K. A.; Daldal, F.; Rousseau, D. L. J. Am. Chem. Soc. 1995, 117, 9363-9364. (9) Wang, J.; Ching, Y.-c; Rousseau, D. L; Hill, J. J.; Rumbley, J.; Gennis, R. B. J. Am. Chem. Soc. 1995, 115, 3390-3391. (10) Wang, J.; Takahashi, S.; Rousseau, D. L. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9402-9406. (11) Wang, J.; Takahashi, S.; Hosler, J. P.; Mitchell, D. M.; FergusonMiller, S.; Gennis, R. B.; Rousseau, D. L. Biochemistry 1995, 34, 98199825. (12) Hirota, S.; Ogura, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Nagai, M.; Kitagawa, T. J. Phys. Chem. 1994, 98, 6652-6660. (13) Hosler, J. P.; Kim, Y.; Shapleigh, J.; Gennis, R.; Alben, J.; Ferguson-Miller, S.; Babcock, G. T. J. Am. Chem. Soc. 1994, 116, 55155516. (14) (a) Findsen, E. W.; Centeno, J. A.; Babcock, G. T.; Ondrias, M. J. Am. Chem. Soc. 1987, 109, 5367-5372. (b) Schelvis, H.; Varotsis, C.; Deinum, G.; Ferguson-Miller. S.; Babcock, G. T. J. Am. Chem. Soc. 1997, 119, 8409-8416. (15) Varotsis, C.; Kreszowski, D. H.; Babcock, G. T. Biospectroscopy 1996, 2, 331-338. (16) Alben, J. O.; Caughey, W. S. Biochemistry 1968, 7, 175-183.

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