Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme.

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.

Spectral Characterization of Diarylpropane Oxygenase, a Novel Peroxide-dependent, Lignin-degrading Heme Enzyme* (Received for publication, October 12, 1984) Laura A. Andersson, V. Renganathan Diarylpropane oxygenase, an HzOZ-dependent lignin-degrading enzyme from the basidiomycete fungus Phunerocliuete chrysocsporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (Oz). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at -420 nm, as in ferrous40 myoglobin (Mb), and not like the -450 nm absorption of the CO complex of the ubiquitous heme monmxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural ~~u i r e m e n t s for heme ironbased oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation.
The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin-and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, h e x a c~r d i n a t e ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(I1) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.
Lignin is a complex, optically inactive, heterogeneous, and random biopolymer (1) that comprises 20-30% of woody plants. Thus, its catabolism and utilization as a renewable resource are of great interest. A variety of white rot basidiomycetous fungi are capable of catabolizing lignin (2) in an apparently nonspecific process (3-7) which has been shown by both chemical and physiological studies to be oxidative (8)(9)(10)(11)(12)(13). Recent studies have revealed that Phanerochaete chrysosporiun, a white rot basidiomycete, produces an extracellular, H~O*-dependent, Iigninolytic enzyme when cultured under aerobic conditions (14, 15). Enzyme activity is absent both from nonligninol~ic cultures and from cultures of a nonligninolytic mutant of this organism (14, 16,17).
To more fully characterize this novel H20B-dependent heme enzyme, we have compared the electronic absorption, electron p a r a m a~e t i c resonance (EPR4), and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known active-site structure. Specifically, we have sought to determine whether the coordination environment of the diarylpropane oxygenase prosthetic group is most similar to that of the heme monooxygenase, cytochrome P-450, the 02-transport protein myoglobin (Mb), or to catalase and the various peroxidase-type enzymes such as horseradish peroxidase (HRP) and cytochrome c peroxidase.
Our findings show that the optical, RR, and EPR spectral properties of diarylpropane oxygenase are closest to those of myoglobin and suggest that these two heme proteins have a similar iron environment and coordination geometry. Furthermore, the diarylpropane oxygenase spectral properties differ markedly from those of cytochrome P-450, suggesting both different active-site environments and different enzymatic mechanisms for the two enzymes. Finally, diarylpropane oxygenase is also spectrally distinct from the H20nrequiring enzymes: horseradish peroxidase, cytochrome c peroxidase, and catalase.
The N-dealkylating enzyme, secondary amine monooxygenase from Pseudomonas aminouorans, has also been suggested to be a heme monooxygenase (23, 24). In addition, the enzyme-substrate complex of heme oxygenase, the system that degrades hemin to biliverdin, is spectrally similar to myoglobin and also appears to function as an oxygenase (25).
Under nonphysiological conditions, hemoglobin will hydroxylate aniline, in a reaction that is inhibited by catalase (26).

EXPERIMENTAL PROCEDURES
Diarylpropane oxygenase was isolated and purified from the extracellular medium of agitated, aerobically cultured P. c~~s o s p o~~u~, as described previously (20). Electrophoretically homogeneous diarylpropane oxygenase (20) was concentrated to -200 p M in 20 mM sodium succinate buffer, pH 4.50, 100 mM NaC1. Sperm whale myoglobin (Sigma Type 11) was made to -300 ~L M in H20, dialyzed against ferricyanide to ensure full oxidation, and exchanged into 20 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl. All of the proteins studied herein were compared at their respective pH optima. Resonance Raman spectroscopic studies were performed on diarylpropane oxygenase samples from two different protein p r e p~t i o n s .
No differences could be observed in the RR spectra of the two samples. Complexes of diarylpropane oxygenase and Mb with CNand N i were generated by addition of concentrated stock solutions to a final ligand concentration of 15 and 30 mM, respectively. The ferrous form of the enzyme was prepared under rigorously anaerobic conditions, as described previously (20). Integrity of enzyme samples was confirmed by absorption spectroscopy immediately prior to and following RR spectroscopy. In no case was evidence of laser-induced degradation observed.
Resonance Raman spectra of diarylpropane oxygenase and Mb were obtained on samples maintained at -2 "C in melting point capillaries using a back-scattering geometry from a sample Dewar. Excitation was provided by Spectra-Physics ion lasers (164-05 Ar, 164-01 Kr). The Raman spectrophotometer and computer interface have been reported previously (27). EPR spectra of native diarylpropane oxygenase and aquometMb were obtained at liquid helium temperature on a Varian E-109 Century Series spectrometer equipped with a Varian E-102 microwave bridge. Data were obtained under the following conditions: modulation amplitude, 5 Gauss; microwave power, 0.5 mW; frequency, -9.254 GHz; modulation frequency, 100 KHz. EPR spectra of the cyanide complex of diarylpropane oxygenase were also obtained at liquid helium temperatures on the above instrument. Data were obtained under the following conditions: modulation amplitude, 8-10 Gauss; microwave power, 0.1 mW, frequency, -9.254 GHz; modulation frequency, 100 KHz. EPR g values are calculated from field strengths determined by a Varian Gaussmeter and are estimated to be accurate to kO.01 units.

RESULTS
The optical absorption band positions of ferric, ferrous, and liganded forms of diarylpropane oxygenase, adapted from  Table I. The EPR spectrum of native diarylpropane oxygenase at 4 K is shown in Fig. 1. EPR g values for ferric diarylpropane oxygenase and its cyanide derivative are shown in Table 11 Table 111. The vibrational bands of diarylpropane oxygenase are listed and assigned in Table IV,    1372 cm" support the assignment of a high-spin ferric heme (Tables I11 and IV

DISCUSSION
The three methods chosen for this spectroscopic study of the diarylpropane oxygenase provide complementary information. It is generally accepted that two different heme proteins having identical coordination environments will exhibit wavelength variations no greater than f 5 nm for any given transition in their electronic absorption spectra (45). EPR spectroscopy can provide information on both the spin state and the symmetry of the metal center. For example, among a series of high-spin ferric heme systems that include aquometMb, hemoglobin (Hb), HRP, cytochrome c peroxidase, and catalase, EPR spectra of the oxygen carriers were shown to possess a more axial or tetragonal character as compared with a more rhombic symmetry for the peroxidases and catalase (46), suggesting that the protein environments of the hemes in the peroxidases and catalase are dissimilar  Table I) has features typical of a high-spin ferric heme system. For example, its Soret maximum (407 nm) is within 2.5 nm of that of high-spin hexacoordinate aquometMb and catalase and within 4 nm of that of high-spin pentacoordinate acid HRP; however, the diarylpropane oxygenase Soret band is significantly red-shifted (>lo00 cm") from that of ferric cytochrome P-450 (391 nm). The oxygenase absorption at 500 nm and the high-spin marker band at 632 nm are similar within a narrow 350-cm" spread in all five of these heme proteins; this agreement supports the high-spin ferric assignment of native diarylpropane oxygenase (20). Furthermore, the finding that both p e n~c~r d i n a t e HRP and P-450 have their high-spin marker bands at 2641 nm, whereas those of hexacoordinate aquometMb and diarylpropane oxygenase are at -633 nm, suggests that the native oxygenase may also be hexacoordinate with a loosely bound sixth ligand, such as H20, as found in aquometMb. Contrary to a previous suggestion (19), the ferric diarylpropane oxygenase absorption spectral pattern is distinct from that of low-spin, hexacoordinate cytochrome bs.
The absorption maxima of the cyano and azido adducts of the oxygenase are virtually identical to those of the corresponding low-spin, hexacoordinate metMb derivatives, but do show some distinction from the spectra of the low-spin, hexacoordinate Ng-HRP, CN-SHRP, and CN-. P-450 complexes ( Table I). Unlike catalase, diarylpropane oxygenase can be reduced; the ferrous enzyme has its Soret and visible bands at 435 and 556 nm, respectively, in good agreement with the analogous bands of both high-spin, pentacoordinate deoxy-h4b and ferrous HRP. However, only a poor match is observed between these spectral features and those of ferrous P-450. The bands of the ferrous-CO complex of diarylpropane oxygenase are at 420, 535, and 568 nm and agree fairly well with those of the ferrous-GO complexes of both Mb and HRP, but again are distinctly different from those of ferrous-CO P-450. These observations strongly suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is likely to be a histidine or histidinate as found in Mb and HRP, respectively, Dewar; other conditions as in Fig. 1. B, CN

Soret excitation Visible Svstem excitation Ligands
High-spin Fe(II1) DAPOX" AquometMb" F--metMb'   spin, hexacoordinate aquometMb and confirm the high-spin 11). As mentioned above, a comparison of the g values of ferric assignment ( Table 11). Both of their EPR spectra are catalase and the two peroxidases with those of the Oz-binding quite distinct from those of high-spin pentacoordinate ferric proteins, Mb and Hb, led to suggestions that the heme envicatalase, HRP, cytochrome c peroxidase, and P-450 (Table ronments differed between the two functionally distinct types of heme proteins (47). Thus, the great similarity between the EPR g values of native diarylpropane oxygenase and metMb is also indicative of similar active-site environments in the two systems. The g values for the CN-complex of the oxygenase (Table 11) are in the range observed for other low-spin, hexacoordinated cyano-heme complexes.

Resonance Raman Spectroscopy
Native Diarylpropane Oxygenuse-Resonance Raman spectra of the native enzyme obtained in the high-frequency region (Fig. 2) are also indicative of a high-spin ferric heme. The spin-and oxidation-state marker bands (40) of native diaryl- propane oxygenase (vl0, 1612; v19, 1558; v3, 1479; and v4, 1372 cm") are at frequencies expected for high-spin ferric hemes and are within f 4 cm" of the analogous RR bands of highspin ferric aquometMb (Tables I11 and IV). A comparison of these marker bands with those of other high-spin Fe(II1) porphyrin systems further supports a hexacoordinate structure of the oxygenase heme that agrees with the electronic absorption spectral evidence. For example, v3 in the oxygenase spectra (1479 cm") is within 1-4 cm" of v3 in the spectra of hexacoordinate bis(dimethylsu1foxide)-Fe(I1I)protoporphyrin IX complex (1480 cm") (41) and hexacoordinate F-.metMb (1482 cm") (41). In contrast, pentacoordinate high-spin ferric systems such as chloro-Fe(II1)-protoporphyrin IX (41), HRP (40), catalase (40), and P-450 (40) all have their v3 at -1488-1500 cm", some 9-21 cm" above the diarylpropane oxygenase frequency. The spin-state band, vI9, for the oxygenase (1558 cm") is also within the narrow range (1557-1562 cm") seen for the hexacoordinate heme systems, whereas v19 ranges from -1567 to 1576 cm" for pentacoordinate ferric heme systems. Overall, the general RR characteristics of native diarylpropane oxygenase and, by inference, the active-site environment are very similar to those of aquometMb, but unlike those of cytochrome P-450, HRP, or catalase. These observations again preclude a cysteinate ligand for the oxygenase (as found in P-450) and argue against a tyrosinate fifth ligand (as found in catalase) or a histidinate/strongly hydrogen-bonded histidine (52, 53) ligand (as found in HRP). Resonance Raman spectroscopy has been a particularly useful technique for the identification of tyrosinate coordination in both heme and non-heme iron proteins. A set of four characteristic peaks appears at approximately 1600, 1500, 1275, and 1170 cm" that are enhanced by excitation within the tyrosinate + Fe(II1) charge transfer band (54,55). The unequivocal absence of any such features in the RR spectra of diarylpropane oxygenase almost certainly rules out tyrosinate as a possible axial ligand to the heme group of this enzyme.
Ligated Diarylpropane Oxygenase-The complexes of the ferric diarylpropane oxygenase with CN-and N; have RR spectra typical of low-spin, hexacoordinate ferric heme systems ( Fig. 4; Tables I11 and IV). For example, vl0 is shifted from 1612 cm" in the native form to 1639 cm" in the spectra of CN-and N; adducts; a similar shift is seen when aquometMb is converted to the cyano and azido forms. The frequencies assigned to v19 and v3 are also shifted in the spectra of the low-spin CN--and N;-oxygenase species, from 1558 and 1479 cm" to -1577 and 1500 cm", respectively. These spectral shifts concur with those observed for the conversion of high-spin Fe(II1) to low-spin Fe(II1) heme complexes. The most notable difference in the spectra of the cyano complexes of diarylpropane oxygenase and metMb is that the 1639-cm" feature (vlo) of the former is markedly less intense than the analogous feature in the CN-. metMb spectrum. Whereas RR peak positions are a property of the electronic ground state of a given molecule, RR peak intensities are related to the excited electronic state of the molecule (56). Thus, the decreased intensity of vl0 in the CN--oxygenase RR spectrum may indicate a different excited state for this species than for CN-. metMb. It is unlikely that the weak v10 feature is due to incomplete formation of the low-spin complex, since the other spin-state marker bands (v19 and v3) are in the expected frequency range and have more typical intensity patterns. As was seen with native diarylpropane oxygenase, the RR spectral properties of its CN-and N; derivatives most closely resemble those of the analogous metMb complexes and are again less similar to those of CN-.HRP. This is especially evident for u19, observed at 1577 and 1579 cm" for the oxygenase and metMb .cyano complexes, respectively, but at 1590 cm" for CN--HRP. These findings again suggest that neutral histidine is a most likely candidate for the fifth ligand of diarylpropane oxygenase.
Ferrous Diarylpropane Oxygenase-The RR spectra of the reduced form of the enzyme (Fig. 3) are indicative of a highspin pentacoordinate ferrous iron. The oxidation-state marker, v4, is at 1357 cm", a frequency typical of normal Fe(I1) heme systems, rather than the low-frequency v4 (1346 cm") of ferrous cytochrome P-450 (Table 111). Since the position of v4 in RR spectra of P-450 has been correlated with the electronic properties of its cysteinate ligand (57), we conclude that the axial ligand of ferrous diarylpropane oxygenase is not a cysteine sulfur. The spin-state markers of the reduced oxygenase, u10 (-1603 cm"), u19 (1554 cm"), and v3 (-1470 cm"), are at frequencies expected for high-spin, pentacoordinate ferrous heme systems and differ notably from the analogous bands of low-spin, hexacoordinate bis(imidazole)Fe(II)protoporphyrin IX or cytochrome b5.
In general, the RR band positions of ferrous diarylpropane oxygenase are similar to those of both deoxy"b and ferrous HRP. An interesting exception is observed for the band assigned as v(C=C): 1625 cm" for the oxygenase and 1627 cm" for HRP, but 1618 cm" for deoxy-Mb. Desbois et al. (43) recently reported that the functionally distinct 02-binding proteins and peroxidases were also distinct in their RR spectra. These authors noted that the position of the vinyl C=C stretching mode of the ferrous proteins could distinguish between the two functional systems, being found at 1618-1621 cm" for oxygen carriers and at 1625-1627 cm" for peroxidases. They proposed that this spectral difference arises from an increase in electron density on the vinyl carbons of the protoporphyrin IX prosthetic group in the peroxidases, relative to that in the Op-binding systems (43). However, this raises a question as to whether an increased electron density on the vinyl groups of peroxidases (and possibly diarylpropane oxygenase) serves a functional role. DiNello and Dolphin (58) demonstrated that apo-HRP reconstituted with deuterohemin (iron protoporphyrin 1X with hydrogen atoms in place of the two vinyl substituents) still retains considerable peroxidase activity (-26% of native HRP activity) and that the key substituents for retention of peroxidase activity were the propionic acid side chains.
Application of these observations to the RR spectra of ferrous diarylpropane oxygenase indicates an electronic structural similarity to ferrous HRP and the peroxidases, at least with respect to the vinyl substituents on the common iron protoporphyrin IX prosthetic group. Extension of these ideas to the high-spin ferric systems (Table 111) also places diarylpropane oxygenase among the peroxidases; its v(C=C) at 1627 cm" is somewhat closer to the vinyl frequency of HRP (1632 cm") than to that of aquometMb (1620 cm"). Our previous observations, however, showed that electronic ab-

Spectral Characterization
of Diarylpropane Oxygenase sorption, EPR, and RR spectra of both the native and CN-ligated oxygenase arc: generally more similar to those of the analogous myoglobins than to those of HRP.
Thus, it appears that the heme coordination sphere of diarylpropane oxygenase, including axial ligation and activesite environment, is closest to that of Mb, whereas the vinyl substituents of the enzyme prosthetic group might have the increased electron density proposed for the peroxidases. Since diarylpropane oxygenase functions both as a peroxidase and as an oxygenase but apparently not as an oxygen-carrier, it is then not surprising that this enzyme does not readily fit into any one particular structural category. The diverse chemistry of the enzyme may well be determined by the presence of specific functional domains, as originally conceived by Mason (59, 60).

Structural and Mechanistic Implications
Based upon the results of the three complementary spectroscopic techniques, few, if any, similarities were observed between the properties of HzOz-dependent diarylpropane oxygenase and cytochrome P-450, despite previous speculations (61). In addition, the oxygenase is spectrally and, consequently, structurally distinct from both catalase and HRP. The significance of these findings is 2-fold first, they essentially eliminate the possibility of either a cysteinate or tyrosinate axial ligand to the heme iron of diarylpropane oxygenase; second, they raise questions as to the general mechanism of heme monooxygenase reactions.
The original definition of a monooxygenase is an enzyme which catalyzes the introduction of a single atom of dioxygen into a substrate with concomitant reduction of the second oxygen atom to water (Scheme IV; 21,26). RH + 1 8 0 2 + 2H+ + 2e-.--* R"OH + H2018 SCHEME IV As recently as 1978, cytochrome P-450 was described as the only heme protein known to be normally capable of monooxygenase reactions (22)'~~ and under typical conditions, is not H20a-dependent. The observation that diarylpropane oxygenase can catalyze two types of H,Oz-dependent reactions, the monooxygenase of model compounds with concomitant insertion of one atom of molecular oxygen into the product, and the oxidation of model lignin compounds, renders this heme enzyme of particular interest. Reactions catalyzed by diarylpropane oxygenase include olefinic hydroxylations (14,16), as well as aliphatic diol cleavage and C, oxidations (14)(15)(16)19,20).
It is commonly assumed that the special coordination sphere and environment of the porphyrin iron are directly related to P-450 monooxygenase functions (22,63). P-450 has been convincingly demonstrated to have a cysteine anion as the fifth ligand to the heme iron in its ferric, ferrous, and ferrous-CO states.' This ligand creates an unusually electronrich environment at the heme iron center (33,66) which is believed to be responsible both for the spectral properties of P-450 and for its ability to activate molecular oxygen for insertion into organic molecules. In contrast, respiratory proteins such as myoglobin have histidine as the fifth ligand to the heme iron, whereas horseradish peroxidase has a histidine anion or strongly hydrogen-bonded histidine as its ligand (52, 53). Thus, the observation by Gold et al. (20) that the ferrous-CO complex oE diarylpropane oxygenase has its Soret maxi-See Refs. 24, 64, and 65 for extensive discussions of the evidence for the P-450 coordination environment. mum at -420 nm, a wavelength typical of Mb, Hb, and HRP, but distinct from the -450 nm Soret maximum of ferrous-CO P-450 (22) led us to seek further understanding of the enzyme's oxygenase and oxidation reactions.
Our results, indicating that the oxygenase has a neutral histidine as a fifth ligand, suggest that the mechanisms of oxygen activation and insertion are different for diarylpropane oxygenase and cytochrome P-450. This may be supported by previous studies of lignin degradation (67) which indicate that photosensitizing riboflavin is able to catalyze lignin model compound oxidations similar to those carried out by both the fungus and by the purified enzyme. In addition, "0 is incorporated during the photosensitizing riboflavin reaction (68) in a monooxygenase-like manner. Photosensitizing riboflavin appears to cleave the diarylpropane in a Type I hydrogen abstraction reaction where the radical produced may react subsequently with the ground state oxygen (67).
This mechanism is analogous to that which has been proposed for prostaglandin cyclooxygenase, a heme enzyme which also catalyzes both H20z-dependent oxidations and insertion reactions (69). In one of the proposed mechanistic pathways for prostaglandin cyclooxygenase, a carbon-centered radical is produced, and this radical reacts with molecular oxygen to form a peroxy radical. The latter intermediate ultimately yields the final product via a chain reaction (69). Enzymatic substrate activation followed by reaction of the activated substrate with 0, has also been postulated for the mechanism of protocatechuate oxygenase (70, 71). This mechanism has been supported by several spectroscopic studies (72,73) which indicate that 0, may not bind directly to the Fe(II1) in the enzyme-active site.
The fact that HzO,-dependent diarylpropane oxygenase reacts with H,O, to form a stable peroxy-adduct (19)' suggests that the substrate is oxidized after binding to this adduct. 0 2 may then react with the enzyme-organic substrate complex to form the aldehyde (11) and diol (111) products. Electronic absorption' and RR8 studies of the peroxy-adduct of ferric diarylpropane oxygenase show that it is an Fe(1V) complex, similar to compound I1 of horseradish peroxidase (32). Despite its H202 dependency, the spectral properties of the oxygenase are generally dissimilar to those of horseradish peroxidase, with the exception of the RR frequencies for the vinyl substituents, and are more closely related to those of Mb. Ferrous diarylpropane oxygenase also forms a stable O2 adduct: which is not thought to play a role in the catalytic mechanism. Additional spectroscopic and mechanistic studies are in progress to better define the novel enzymatic capabilities of diarylpropane oxygenase and to correlate these properties with those of other heme proteins and enzymes.