Vol. 269, No. 2, Issue of January
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
14, pp. 1010-1014, 1994 Printed in U.S.A.
Heme-Heme Oxygenase Complex STRUCTURE OF THE CATALYTIC SITE AND ITS IMPLICATION FOR OXYGEN ACTIVATION* (Received for publication, June 29, 1993, and in revised form, August 26, 1993)
Satoshi Takahashi, Jianling Wang, and Denis L. Rousseau From the AT&T Bell Laboratories, Murray Hill, New Jersey 07974
Kazunobu Ishikawa and Tadashi Yoshida From the Department of Biochemistry, Yamagata University School of Medicine, Yamagata 990-23, Japan
Janette R. Host and Masao Ikeda-Saitot From the Department of Physiology and Biophysics.- Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970 "
"
Heme oxygenase, a central monooxygenase enzyme of The enzyme itself is not a hemoprotein, but binds one equivathe heme catabolism and the associated generation of lent of heme resulting in the formation of the heme-enzyme carbon monoxide, forms a 1:l stoichiometric complex complex, which exhibits light absorption spectral properties of with iron protoporphyrin IX,which is a prosthetic ac- hemoproteins (6, 10). tive center and at the same time the substrate of the The presence of two isoforms of heme oxygenase has been enzyme. By using EPR, resonance Raman, and optical established: heme oxygenase-1, which is inducible and highly absorptionspectroscopictechniques, we havedeterexpressed in liver and spleen tissues, and heme oxygenase-2, mined the axial ligand coordination of the enzyme-heme which is constitutive and distributed throughout the body (11). complex. The ferric heme iron in the heme-enzyme comThe two isoforms have differentmolecular masses (-33 kDa for plex at neutralpH is six-coordinate high spin, while at type 1 versus -36 kDa for type 2) and are products of the two alkaline pH(pK, 7.6), the complex becomes low spin. distinctly different genes (12, 13). Amino acid sequence simiSpectra of ferrous forms of the complex indicate that histidine serves as the iron proximal axial ligand and larity between the two isoforms is only about 40%, but there are that the residue is in its neutral imidazole rather than severalstretches of the highly conserved sequences with its imidazolate protonation state. Thus, the active site of matched predicted secondary structure (14). As both isoforms display the same enzymatic activity, it is probable that the active the heme-heme oxygenase complex has a myoglobin-like structure rather than an active site similar to the large site structure, and hence the molecular mechanism of the encytochrome P-450class of monooxygenases.A s a conse- zyme action, is the same between theisoforms as proposed by quence, the activated form ofthe heme-heme oxygenase Rotenberg and Maines(14). The enzyme has a hydrophobic secomplex, a peroxo intermediate, is different from that of quence near its C-terminal end that isinvolved in binding to the cytochromeP-460monooxygenases, in which the ac- microsomal membranes (15).A tryptic digestion of this memtivated form is an oxo intermediate. The overall cata- brane binding C-terminal stretch produces a catalytically aclytic mechanismis probably moreclosely related to thattive 28-kDa water-soluble form, which is a better alternative of othermonooxygenaseswithmyoglobin-likeactive for the structural studies than the full-length enzyme because sites, such as secondary amine monooxygenase. of the relative easeof handling water-soluble proteins (16). Despite its physiological significance, the understanding of the molecular structure of heme oxygenase is severely limited, Carbon monoxide, a stable diatomic gaseousmolecule, is now in part due to the difficulty in obtaining a large amountof the recognized as a physiologic regulator of cGMP like nitric oxide enzyme necessary for structural studies. As a first step tounby activating soluble guanylyl cyclase (1, 2). Carbon monoxide derstand the molecular mechanism of the enzyme action of is formed by the action of the enzyme hemeoxygenase, which heme oxygenase, it is essential to determine the axial ligand has been known as the centralenzyme of heme catabolism (3). coordination structure of the heme iron in the enzyme-heme The enzyme utilizes NADPH and molecular oxygen to catalyze complex. Recent success in the construction of the bacterial the formationof biliverdin and carbon monoxide from iron pro- system overexpressing a 30-kDa soluble form of rat heme oxytoporphyrin M (heme hereafter) through consecutive mono- genase-1 now allows us to carry out structural studies of the oxygenase catalysis steps,which carry outa regiospecific open- enzyme (17). Herein, we report for the first time the axial ing of the tetrapyrrolemacrocyle at the a-mesoposition (4-9). ligand coordination structure of the heme iron in the hemeenzyme complex using the soluble form of recombinant rat * This workwas partially supported by Research Grants GM48714 (to heme oxygenase-1. D. L. R.) and GM39492 (to M. I.-S.)from the National Institute of GeneralMedicalSciences and Grants 02680150 (to T. Y.) from the EXPERIMENTALPROCEDURES Ministry of Education, Science, and Culture, Japan. The purchaseof the Bruker EPR spectrometer system was in part supported by a Grant The enzymatically active water soluble form of the rat heme oxygenRR05659 fromthe National Center for Research Resources. The costs of ase isoform-130-kDa protein, which lacks the 26-amino acid C-terminal publication of this article were defrayed inpart by the payment of page hydrophobic membrane-bound segment with Ser-262 Arg and Sercharges. Thisarticle must thereforebe hereby marked "advertisement" 263 Leu mutations, was expressed in JM109 as described previously in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Dept. of Physiology (17). After bacterial lysis bylysozyme, the enzyme was purified by and Biophysics, Case Western Reserve University School of Medicine, following the procedure reported forthe trypsin digest fragmentof the Cleveland, OH 44106-4970. Tel.: 216-368-3178;Fax: 216-368-5586; E- rat isoform-1, which included ammoniumsulfate fractionation,gel fib tration on Sephadex G75,DEAE-cellulosechromatography,and hymail:
[email protected].
-
"-f
1010
1011
Heme-Heme Oxygenase Complex
r PH
0.8
0.6 a2
8 e w
9
6.0 6.4 7.1
7.5 7.7 7.9 8.4 9.1
9.9
6
7
8
9
1
0
pH
0.4
~ 2 . 6 7g2.21
0.2
. 0 0 400
500
600
700
Wavelength (nm)
FIG. 1.Light absorption spectra of the ferric heme-heme oxygenase complexbetween pH 6 and 10 at 20 "C. The pH values of the sample are listed in the figure. Inset, the fraction of the alkaline form calculated from the pH-dependent changes in the absorbance at 404 nm. The symbols are experimental values, and the curue is drawn by a least-squares fitting to the n = 1 Hendersen-Hasselback equation.
. 0.1
.
0.2
.
0.3
~1.79 .
. 0.4
.
B~ (Tesla)
FIG.2. EPR spectra of ferric heme-heme oxygenase complex measured at 6 K. Measurements were carried out with an incident microwave power of 2 milliwatts and 0.1 millitesla field modulation at M phosphate, 100 kHz.The heme-enzyme complex was dissolved in 0.1 pH 7, or 0. 1 M Tris buffer at pH 8 and 9.
nate high spin ferric hemoproteins (21, 23).2 Hence, the EPR result is in support of the assignment of six-coordinate high spin species in the acidic form of the enzyme-heme complex. When pH is increased from 7 to 9, the amplitude of the high droxyapatite chromatography (16).' The soluble form of heme oxygen- spin EPR signal of the acidic form is reduced and a new low ase was titrated by hemin until 90% of the enzyme was complexed with spin EPR signal withgl = 2.67,g2 = 2.21, andg3 = 1.79 appears. hemin. This water-soluble 30 kDa formof the enzyme retains theheme Resonance Raman spectra of the ferric enzymeconfirmed the oxygenase activity for the conversion of hemin to biliverdin as the pH-dependent spin changesdeduced from the optical and EPR full-length 33 kDa enzyme (17). Light absorption spectra were recorded on a Hitachi U3210 spectro- measurements. In theregion of the Raman spectrumcontainphotometer at 20 "C. X-band EPR spectra were obtained with a Bruker ing the porphyrin skeletal modes, several lines have frequenEPS-300 spectrometer with an Oxford liquid helium cryostat. The fer- cies characteristic of the heme iron spin and coordination states rous nitric oxide complex was generated by the use of Na15N02and (26-28). Specifically the linesassigned as vz and v3 are located sodium dithionite (18). To obtain the resonance Raman spectra, 50 p~ in the regions of 1580-1590 and 1500-1510 cm" for six-coorsamples of the heme-heme oxygenase complex were sealed in a rotating dinate low spin iron, 1560-65 and 1475-85 cm-' for six-coorcell. The laser excitation wavelength was 406.7 nm for the femc form of the enzyme complex and 441.6 nm for the dithionite-reduced ferrous dinate high spin iron, and 1570-1575 and 1490-1500 cm" for ligand-free form. The scattered light was dispersed by a Spex 1.25-m five-coordinate high spin iron,respectively. As shown in Fig. 3, spectrograph and detected with a Photometrics CCD camera. 'Qpically in heme oxygenase at pH 6.5 we find vz and v3 at 1563 and 1482 several 1-minspectra were obtained and averaged to improvethe signal cm", respectively, characteristic of a six-coordinate high spin to noise ratio. The frequencies of the Raman-shifted lines were caliiron atom, and at pH 9 the two modes are at 1581 and 1503 brated against an indene standard. cm-', respectively, characteristic of six-coordinate low spin. The observations from these three different techniques lead RESULTS AND DISCUSSION to the clearconclusion that the heme iron in the ferric hemeFig. 1illustrates the pH-dependent changes in the light absorption spectrum of the ferric heme-enzyme complex between enzyme complex is predominantly in a six-coordinate high spin pH 6 and 10. At pH 6, theenzyme has a Soret peak at 404 nm and a six-coordinate low spin state under acidic and alkaline visible region. As reported conditions, respectively. In comparison to other oxygenases, and bands at 500 and 631 nm in the previously (6, 101, the spectrum is similar to that of methemo- these are uniquecharacteristics. For example, most substrateglobin (19). The Soretabsorption band is known to be sensitive free cytochrome P-450s are six-coordinate low spin (29) and do to the coordination structure of the ferric high spin hemopro- not display an acididalkaline transition.Cytochrome c peroxiteins (20-23). On the basis of the Soret peak position and its dase and horseradish peroxidase are five-coordinate high spin extinction coefficient (165 m"l cm-l at pH 61,the ferric iron in a t acidic and neutralpH (28,301. The hemeiron in indoleamine the heme-enzyme complex at pH 6 is postulated to be six- 2,3-dioxygenase is in a thermal mixture of low spin and high coordinate high spin. At alkaline pH thehigh spin spectrumis spin states which is pH-independent (31); substrate-free Lreplaced by a spectrum with bands at 413, 540, and 575 nm, tryptophan 2,3-dioxygenase becomes mixed spin at alkaline which is similar to those of the low spin form of the hydroxide conditions (32); andsecondary amine monooxygenase only parcomplex of methemoglobin. This pH-dependent spectral change tially converts to low spin at high pH (33). The pH dependence is reversible between pH 6 and 10, and the pK, value of the of the ferric form of heme-heme oxygenase complex is most similar to the oxygen transport and storage molecules: hemochange is estimated to be 7.6 as shown in the inset of Fig. 1. EPR spectraof the ferricheme-enzyme complex are shown in globins and myoglobins. Most of these proteins also have an Fig. 2. The acid form of the ferric heme-enzyme complex exhib- acididalkaline transition at which they convert from six-coorits an EPR spectrum of typical highspin ferric hemoproteins(g dinate high spin to six-coordinate low spin, although often = 6 and g = 2). The high spin signal of the acidic form of the these proteinsdisplay thermal mixtures of spin states (3435). Based on the similarity of the optical absorption, EPR, and enzyme-heme complex is axially symmetric, which is different from rhombic high spin signals often observed for five-coordi- resonance Raman spectra of the heme-heme oxygenase com-
' M. Ito, K. Ishikawa, M. Sato, and T. Yoshida, submitted for publi-
cation.
Some six-coordinatehigh spin hemoproteins with water as a ligand also exhibit rhombic highspin EPR signals as seen in myeloperoxidase and lactoperoxidase (24, 25).
Heme-Heme Oxygenase Complex
1012 Heme-heme oxygenase complex
(Fej+)
406.7nrn excitation
g
g =2.086
'I
0.33
0.32
0.31
=z.m '%(NO):
A = 2.6 mT
'%(His): A = 0.74 mT
0.34
0.35
Cr-ld
1200
I400
1600
FIG.4. EPR spectrum of the ferrous heme-heme oxygenase l6N0complex recorded at 30 K with a microwave power of0.2 milliwatt and 0.1 millitesla field modulation at 100 kHz. The second derivative displayis also presented for clear identificationof the hyperfine couplings.
Raman Shift (crn-1) RG.3.Resonance Raman spectra of the ferric heme-heme oxyg e m e complex at pH 6.5 (A) and 9.0 ( B ) . The laser excitation that water is the sixth ligand (high spin) at neutral pH. wavelength was 406.7 nm.The spin and coordination sensitive lines, v, Acid-base transitions observed in ferric hemoproteins with a and vz shift from 1482 to 1503 cm-' andfrom 1563 to 1581 cm", respectively, at the acidmase transition. water ligand are considered to be linked to the ionization of a distal amino acid residue that forms a hydrogen bondwith the plex at neutral pH to corresponding spectra from the other bound water ligand; the deprotonation of the distal residue, ferric heme proteins which are six-coordinate high spin with histidine in most cases, causes the ionization of the iron-bound bound water, we infer that water is the sixth ligand in heme- water resulting in a predominantly low spin hydroxide form heme oxygenase complexas well. One may argue that thesix- (19). The pK, for the acidhase transition in various hemoprocoordinate high spin state isrealized by the coordination of an teins hasa wide range of values. The observed PK, of 7.6 in the amino acid side chain. There are two well established cases ferric heme-heme oxygenase complex,although low compared where an amino acid residue serves as the sixth ligand with to that in many heme proteins, is consistent with deprotonation proximalimidazole ligation. Coordination of carboxylate or of histidine, as in most of the globins (19), or glutamine as in phenolate to the sixth position of the heme iron could generate the myoglobin distal histidine + glutamine mutant (23). ferric high spin species, as reported for the mutant myoglobin Among the 3 histidine residues conserved in the known heme molecules with the His(E7) Tyr (36) or Val(E11) + Glu (37) oxygenase sequences (39), mutagenesis studies of isoform-1 substitution, and for the abnormal ferric subunits in hemoglo- (17) have shown His-25 is essential for catalytic activity. Anbin M Saskatoon (His(PE7)-+ T y r ) and Milwaukee (Val(PE11) other histidine, His-152 in isoform-2, whichcorresponds to His132 in the isoform-1 sequence, was found to be essential in + Glu) (38). These mutant globins exhibit EPR spectra with rhombic symmetry, different from the axial EPR spectrum of isoform-2 (40), but in preliminary studies on isoform-1 it was heme-heme oxygenasecomplex. The light absorption spectra of not found to be essential.' Additional studies are under way to the mutants showed the so called charge transfer band a t 600 clarify this difference in the isoforms. Ifonly 1 histidine is nm (His(E7) T y r ) and at -620 nm (Val(E11) Glu), to be essential in heme oxygenase, it must form the proximal ligand different from heme-heme oxygenase complex (631 nm). Fur- to the iron (see below); therefore, we cannot identify the distal thermore, a pH-dependent conversion to a low spin species was residue at present. However, the binding of both water and not observed in these mutants. We think that Tyr or Glu liga- hydroxide in theferric heme-enzyme complex and the low pK, tion at the sixthcoordination position of the heme iron in heme- of the acidlbase transition, as compared to other heme proteins, suggest that the distal pocket is very polar. To determine the heme oxygenase complexis rendered unlikely. The low spin EPR spectrum of the alkaline form of heme- proximal ligand, we examined ferrous forms of the heme-enheme oxygenase complex differs from those of the low spin zyme complex. The nitric oxide (15NO) complex of the ferrous heme oxygenhydroxide forms of ferric hemoglobin or myoglobin (gl= 2.59, gz = 2.16, and g3= 1.841, but it is similar to that of the low spin ase-heme complex shownin Fig. 4 exhibits an EPR spectrum of component of the alkaline form of the substrate-bound trypto- NO hemoproteins with a rhombic symmetry (gl = 2.086, g, = phan 2,3-dioxygenase (gl= 2.67, g, = 2.20, and g3 = 1.80) (32) 2.008, and g3 = 1.986). Experiments using 14N0 (data not in which hydroxide was reported as the sixth ligand of the shown) demonstrated that thedoublet with a coupling constant heme iron (31) and cytochrome c peroxidase (gl = 2.69, g2 = of 2.6 millitesla associated with the g, signal in the16N0 EPR 2.19, andg, = 1.80)in which a hydroxide has also been cited as spectrum shown in Fig. 4 is due to the Z = 1/2 of the 16N of the a possible sixth ligand (21). To determine if a hydroxide is the bound 15N0. Thus, the triplet hyperfine splitting with a cousixth ligand in the alkaline form of the heme-heme oxygenase pling constant of 0.74 millitesla is associated with the Z = 114N complex, we have measured the resonance Raman spectrum of nucleus of the axial ligand trans to the bound NO (18,41). This the alkaline form of the heme-enzyme complex in thepresence firmly establishes that the axial heme ligand of the enzymeof H2160and H21s0.3 In these measurements, we were able to heme complex is a nitrogenous base, likely an imidazole group identify an Fe-160H stretching mode at 546 ax-', thereby con- of histidine. This assignment was refined by the resonance firming the hydroxide ligand in the alkaline form of the heme- Raman spectrum of the ligand-free ferrous heme enzymecomheme oxygenase complex. This fmding supports our suggestion plex (Fig. 5), where a line is detected at 218 ax", a frequency characteristic of an iron-histidine stretching mode in five-coorS. Takahashi, J. Wang, D. L. Ruusseau, K. Ishikawa, T.Yoshida, J. dinate ferrous hemoproteins (42). Thus, the current spectroscopic results, which demonstrated the presence of a proximal R. Host, and M.Ikeda-Saito, submitted for publication. -+
-+
-+
Heme-Heme Oxygenase Complex
200
400
600
Raman Shift (cm-1) FIG.5. Resonance Raman spectrum of the ferrous ligand-free heme-heme oxygenase complex in 0.1 M phosphate bder, pH 8.6. The line at 218 cm” is assigned as the iron-histidine stretching mode. The laser excitation wavelength was 441.6 n m .
histidine residue in the enzyme, are in accordance with the site-directed mutagenesis experiments in which at least one functionally essential histidine residue was postulated. In terms of g-values and hyperfine coupling constants, the EPR spectrum of the heme-heme oxygenase NO complex is similar to that of myoglobin NO complex rather thanthose of peroxidase NO complexes (41), suggesting that the proximal histidine in the heme-heme oxygenase complex is a neutral imidazole as in myoglobin rather than an imidazolate as in peroxidase enzymes. The resonance Raman spectnun of the heme-heme oxygenasecomplex, in which the iron-histidine stretching mode is located at 218 cm-l, confirms this assignment since the imidazolate-iron stretching mode is in the240 cm-’ region whereas for the neutral imidazole-iron complex the stretching mode is in the200-220 cm-’ region (42). In the catalytic cycle of heme-containing monooxygenases, the presence of a peroxidase compound I type of structure (high-valent iron oxo complex) has been postulated as an essential intermediate (43). On the basis of spectroscopic and structural information on the heme-containing oxygenases and peroxidases (43-45), a “push-pull” mechanism has been proposed where the generation of the high valent iron-oxo intermediate, which requires the cleavage of the 0-0bond of the iron bound peroxide moiety, is facilitated by an anionic proximal ligand and/or polar distal residues. In the cytochrome P-450 group of enzymes, the thiolate axial ligand, with its enhanced basicity, facilitates cleavage of the bond byserving as a strong internal electron donor. In cytochrome c peroxidase and horseradish peroxidase, the proximal axial ligand is a histidine with imidazolate character which stabilizes the higher valence state. At the same time, the polar environment of the distal pocket by histidine and arginine residues further contribute to the stabilization of the high valent oxo intermediates. The heme pocket structure of the heme-heme oxygenase complex, which has a neutral imidazole as its proximal ligand, is clearly different from that of cytochrome P-450s and peroxidases. Thus, the mechanism of oxygen activation in heme oxygenase is expected to be different from these other enzymes. Indeed, based on the generation of hydrogen peroxide by the heme-heme oxygenase complex, Noguchi et al. (46) proposed that a “peroxo”intermediate isthe reactive form of the enzyme. These ideas were confirmedby Wilks and Ortiz de Montellano (471, who found that thereaction of the heme-heme oxygenase complex with ethyl hydroperoxide yieldedan ethoxy porphyrin derivatives ruling out the involvement of a ferry1 intermediate in thehydroxylation step.
1013
The myoglobin-like pocket of heme oxygenase places it in a class of a few other mono- and dioxygenases that are also coordinated to a neutral imidazole. These include the monooxygenase, Pseudomonas aminovomns secondary amine monooxygenases (33),and the dioxygenases, L-tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (31). Furthermore, hemoglobin and myoglobin may function as oxygenases (48) and even as heme oxygenases (49). The spin equilibria of the heme iron in the femc forms of each of these proteins and enzymes and the PK, values of their acidhase transition are different, so the tertiary structure of their heme pocket are clearly not the same. Further studieswill clarify the role played by the structure of the heme pocket residues in the catalytic function of these hemoproteins and in which of them the catalytically active form is a peroxo intermediate as in the hemeheme oxygenase complex. REFERENCES 1 Brune, B., and Ullrich, V. (1987)Mol. Phurmacol. 32,497404 2 Verma, A,, Hirsch, D. J., Glatt, C. E.,Ronnett, G. V., and Snyder, S. H. (1993) Science 26S, 381384 3 Maines, M. D. (1988)FMEB J . 2,2557-2568 4 Tenhunen, R.,Marver, H. S., and Schmid, R. (1969)J. Bwl. Chem. 244,63& 6394 5. Brown, S. B., and King, R. E G . J. (1976)Biochem. Soc. ’?Fans. 4,197-201 6 Yoshida, T.,and Kikuchi, G . (1978)J. Biol. Chem. 269,4224-4229 7,Yoshida, T., and Kikuchi, G . (1978)J. Biol. Chem. 253,4230-4236 8 Yoshinaga, T.,Sassa, S., and Kappas, A. (1982)J. Bwl. Chem. 257.7778-7785 9 Yoishida, T.,Noguchi, M., and Kikuchi, G. (1982)J. Biol. Chem. 257,93459348 10.Yoshida, T.,and Kikuchi, G . (1979)J. Bwl. Chem. 254,44874491 11. Maines, M. D., Trakshel, G . M., and Kutty, R. K (1986)J. Bid. Chem. 261, 411-419 12 Miiller, R. M., Taguchi, H., and Shibahara, S. (1987)J . Biol. Chem. 262, 6795-6802 13. McCoubrey, W.K , Jr., Ewing, J. F, and Maines, M. D. (1992)Arch.Biochem. Bwphys. 29413-20 14. Rotenberg, M. O., and Maines, M. D. (1991)Arch. Biochem. Biophys. 2S0, 336-344 15 Yoshida, T.,and Sato, M. (1989)Biochem. Biophys. Res. Commun. 169,1086 1092 16. Yoshida, T.,Ishikawa, K , and Sato, M. (1991)Eur. J. Biochem. I@& 729-733 17. Ishikawa, K , Sato, M., Ito. M., and Yoshida, T. (1992)Biochem. Bwphys. . . Res. Commun. 182,981-986 18. Hori, H., Ikeda-Saito, M., and Yonetani, T.(1981)J. Biol. Chem. 266.784% 7855 . .”
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027-1036 hem.
1014
Complex
OxygenaseHeme-Heme
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47. Wilks, A., and ortiz de Montellano, P. (1993)J . Inow. Biochem. 61,269 48. Mieyal, J. J., Ackerman, R. S., Blumer, J. L., andFreeman, L.S. (1976)J . Biol. Chem. 251,34363441 49. O'Carra, P. (1975)in Porphyrins and Metalloporphyrins (Smith, K. M., ed) pp. 123-153, Elsevier Science Publishers B,V, Amsterdam
