PRELIMINARY COMMUNICATION

J. Biochem., 80, 1447-1451 (1976)

P-450cam in the Ferrous High-spin State Yukihiro OZAKI,* Teizo KITAGAWA,* Yoshimasa KYOGOKU,* Hideo SfflMADA,** Tetsutaro IIZUKA,** and Yuzuru ISfflMURA** •Institute for Protein Research, Osaka University, Suita, Osaka 565, and "Department of Biochemistry, School of Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo 160 Received for publication, September 21, 1976

Resonance Raman spectra of cytochrome P^50 e i m (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm"1) in comparison with those of other reduced hemoproteins (—1355—1365 cm"1), whereas that of oxidized P-450clm was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450c»m can be explained by assuming electron delocalization from the fifth ligand, presumably a thioIate anion, to the antibonding n orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm"1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450CEm to P-420. The Raman spectrum of reduced P-450Cam-metyrapone complex was very similar to that of ferrous cytochrome bt.

Cytochrome P-450 (P-450) is a protoheme-containing monooxygenase functional in the metabolism of various substances such as steroids and xenobiotics (/). The enzymatically active form of this enzyme yields a Soret band near 450 nm upon formation of the CO adduct of its reduced form (P-450-CO), whereas the inactive form (P-420-CO) gives a band near 420 nm. The origin of the anomalous Soret spectra of P-450-CO has been ascribed to the coordination of a thiolate anion as the fifth ligand Abbreviations: cytochrome P-450c»m, cytochrome P450 from Pseudomonas putida grown on camphor; P-450c»m-Mp, metyrapone complex of P-450c»m; Mb, myoglobin. Vol. 80, No. 6, 1976

of the ferrous heme iron, based on model compound studies (2-5). MCD studies of P-450-CO supported the above conclusion (6-8). The EPR spectrum of P-450 without substrate (Fe*+ low spin), characterized by a set of three g values near 2.4, 2.2, and 1.9 (9), has been approximately reproduced by that of iron-porphyrin with an arbitrary N base and a thiolate anion as axial ligands of the heme iron, suggesting the coordination of a thiolate anion in ferric P-450 (10-12). Resonance Raman scattering from hemoproteins has been shown to reveal the vibrational spectra of iron-porphyrin surrounded by an apoprotein. Raman intensities and frequencies for some of the porphyrin ring modes apparently

1447

Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1447/855715 by Rudolph Matas Medical Library user on 17 January 2019

An Anomaly in the Resonance Raman Spectra of Cytochrome

1448

The P-450Cim was purified from Pseudomonas putida [ATCC 17453] grown on camphor by a method similar to that described by Peterson (16). The details will be described elsewhere (17). The purity of the P-450 c a m , estimated from the ratio of absorbances at 392 nm/280 nm (18), was 1.1. The concentration of P-450 cam was determined spectrophotometrically using e ^ = 8 7 . 0 for the high-spin ferric form (19). P-420 was prepared by the treatment of ca. 0.1 mM P-450 cam solution with 23 % V/V acetone at room temperature in the absence of D-camphor according to the method of Yu and Gunsalus (20). For the measurement of Raman spectra, 0.1 mM P-450 cam in potassium phosphate buffer (50 mM, pH 7.4) with or without D-camphor was placed in a cylindrical cell. After each measurement, the CO adduct of the sample used for Raman scattering was transferred into a microcell ( 1 x 3 x 3 0 m m 3 ) and the absorption spectrum was measured with a Hitachi 124 spectrophotometer. Raman spectra were excited with the 488.0 nm line of an argon laser and were recorded on a JEOL 400D Raman spectrometer. Frequency calibration was performed with indene (21), yielding an accuracy of ± 2 cm" 1 for the present measurements. Other experimental details were described elsewhere (22). The resonance Raman spectrum of reduced P-450 cam in the presence of D-camphor is compared with that of deoxy Mb in Fig. 1A. The frequencies of Raman lines at 1604 and 1467 cm" 1 of reduced P-450 c a m suggested that the state of heme was ferrous high-spin (15), although the latter line was

P45Ocom (reduced)'

(A)

deoxy Mb

P450cafnMP (reduced) ~~

(B)

cyt bjdi)

Fig. 1. (A) Resonance Raman spectrum of reduced P-45Oc»m in the presence of D-camphor and that of deoxy Mb. (B) Resonance Raman spectrum of reduced P-450c.nu- metyrapone complex and that of ferrous cytochrome bs. Instrument conditions: laser, 488.0 nm line (Spectra Physics, model 164); power, 200 mW at laser output; slit width, 3 cm"1; time constant 3.2 sec; scan speed, 25 enr'/min; sample temperature, 10°. 5 cm" 1 lower and more intense than the 1472 cm" 1 line of deoxy Mb. The intensity ratio of the two Raman lines at 1588 and 1562 cm" 1 also supported the above interpretation (13). A pronounced difference between the resonance Raman spectra of reduced P-450Cam and deoxy Mb was found in the frequency of the so-called "oxidation-state marker" (hereafter referred to as Band IV, according to Ref. 75). Usually Band IV is located between 1355 and 1365 cm" 1 for ferrous and between 1370 and 1375 cm" 1 for ferric hemoproteins (Table I). Indeed, Band IV of deoxy Mb appeared at 1355 cm" 1 , whereas no Raman line was observed between 1350 and 1380 cm" 1 for the reduced P-450 c a m . Instead, an intense Raman line was observed in a lower frequency region (1346 cm" 1 ). The 1346 cm" 1 line was polarized (depolarization ratio=0.2) as was the 1355 cm" 1 line of deoxy / . Biochem.

Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1447/855715 by Rudolph Matas Medical Library user on 17 January 2019

depend upon the spin and oxidation states of the heme iron (13,14). Furthermore, it was pointed out recently that the resonance Raman spectra may distinguish between two types of ferrous low-spin hemoproteins specified in terms of the binding mechanisms of axial ligands to the heme iron, namely, a and ir types of binding (15). In the present study, we report the resonance Raman spectra of Pseudomonas putida P-450 (P-450Oam) and of its inactive form, P-420, in comparison with those of other protoheme-proteins such as myoglobin (Mb) and cytochrome bb (cyt b6). An anomaly was found in the resonance Raman spectrum of reduced P-450 oam but not in those of reduced P-420, reduced metyrapone complex of P-450 cam (P-450Oam-Mp), and several complexes of oxidized P ^ 5 0 e a m .

PRELIMINARY COMMUNICATION

RESONANCE RAMAN SPECTRA OF

1449

TABLE I. The frequencies of Band IV in the resonance Raman spectra of hemoproteins and iron-porphyrin complexes [cm"1].

deoxy Mb cyt c cyt c, cyt/ cyt c'

Reduced

1,375

1,361

MbCO HbCO HbO, HbCNQH,

1,355 1,363 1,359 1,361 1,355*

Heme a Heme a (Im), Fe(OEP)Cl Fe(OEP)Im3

1,371 1,371 1,374

Oxidized

Reduced 1,377 1,373 1,373 1,373

1,375

1,372 1,373 1,373

l,372i

FeProto(CN)j FeProto{Im),

1,375" 1,375b

1,360"

1,374 1,374 1,373 1,374

1,360 1,359

1

Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1447/855715 by Rudolph Matas Medical Library user on 17 January 2019

cyt b, MbHjO MbF MbCN Mblm

Oxidized

Ref. 26 " Kitagawa, T., Kyogoku, Y., & Orii, Y., to be published. Other frequencies were taken from Refs. 14 and 20. Abbreviations: cyt c, horse heart cytochrome c; cyt ca, Desulfovibrio vulgaris cytochrome c,; cyt /, Spirulina platensis cytochrome / ; cyt c', Rhodopseudomonas palustris cytochrome c', OEP, Octaethylporphyrin; Proto, Protoporphyrin.

Mb. Although ferrous protoheme-proteins have been shown to exhibit a weak, anomalously polarized line near 1340 cm" 1 (22), the 1346 cm" 1 line of reduced P-450Oam is clearly different from this because of the different polarization features. Therefore it can be concluded that the 1346 cm" 1 line of reduced P-450 cam corresponds to the 1355 cm" 1 line (Band IV) of deoxy Mb. The frequency of Band IV of microsomal P-450 in the reduced state (1341 cm" 1 ) was also lower than that of deoxy Mb (25). When metyrapone was bound to the sixth coordination position of reduced P-450Cim, the resulting resonance Raman spectrum appeared to be closely similar to that of ferrous cyt-66, as shown in Fig. IB, where two histidyl imidazoles are coordinated to the heme iron (24). It is noteworthy that the resonance Raman spectrum of P-450 ci . m • Mp was of typical ferrous low-spin type, in which two axial ligands are bound to the heme iron through a

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measur...
291KB Sizes 0 Downloads 0 Views