Resonance raman studies of models for the reduced states of cytochrome P 450

Abstract

The catalytic cycle of cytochrome P 450 includes four stable states: a low spin ferric resting state A, a substrate bound high spin ferric state B, a high spin ferrous state C and a low spin ferrous oxy state D. When the system in the state C is exposed to carbon monoxide instead of oxygen a low spin ferrous carboxy state D′ is generated. Several models have been proposed for states B, C, D′ and recently state D [1]. A large variety of spectroscopic techniques have been used to probe the similarities between the model compounds and the actual enzymatic states. Resonance Raman spectroscopy (RR) is a very sensitive technique to investigate specifically the active site of hemoproteins. RR spectra have been obtained for Cyt P 450 from various origins [2–4]: when compared to those of other hemoproteins, the frequencies of the so-called ‘oxidation state’ marker band [5] are unusually low in states C and D′; ‘spin marker bands’ frequencies [5] have been used to monitor the coordination of the iron atom in the ferric states A and B. Recently the FeS stretching mode has been detected at 351 cm −1 in oxidized Cyt P 450 CAM [6]. We report here the results of a RR study of [Fe II(T pivPP)(X −)(L)]18C6Na +* complexes, as models for states C, D′ and D (an investigation of a state A model has already been published [7]). Using the nomenclature propsed in [8], the main RR frequencies are given in Table 1. t001 RR Frequencies (cm −1) of [Fe II(T pivPP)(X −)(L)]Na +18C6 Complexes. Complex Porphyrin vibration FeL vibr. X − L A B C D C 6HF 4S − 1341 a a a 369 Cl − 1343 1355 1494 1545 369 OH − 1344 1355 a a 371 C 6HF 4O − 1343 1354 a 1545 369 C 6HF 4S − CO 1364 a 1567 380 479 C 6HF 4S − O 2 1366 a a 379 a Not observed All the pentacoordinated ferrous species exhibit very similar porphyrinic frequencies. They compare well with the frequencies of the typical high spin ferrous complex Fe(TPP)(2-Me Im) (A = 1345, B = 1361, C = 1500 and D = 1538 [8]). Moreover the A frequency of the carboxy adduct is very close to that of Fe(TPP)(py)(CO) [12], whereas that of the oxy adduct is the same as that of Fe(T pivPP)(1-Me Im)(O 2) [9]. Therefore our RR data do not stress any special π donor properties of the RS − ligand that would induce an extra lowering of the oxidation state marker band frequencies. Soret excitation of the low frequency RR spectrum is readily accessible for the carboxy adduct λ max Soret 448 nm, λ exc 454,5 nm: it reveals a new strong polarized band at 479 cm −1. The intensity of this band decreases with partial photodissociation of the CO ligand. An isotopic substitution experiment, using 13CO, induces a 5 cm −1 lowering of its frequency. This is in good agreement with a calculated shift of −5 cm −1 for the stretching vibration of the FeCO moiety, using the harmonic oscillator approximation. This leads to the assignment of this band to the FeCO stretching vibration. This value is to be compared to those observed for MbCO, HbCO [10] and P 450CO (work in progress).