The active sites of chloroperoxidase and cytochrome P-450-CAM: Comparative spectroscopic and ligand binding properties

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Spectral similarities between chloroperoxidase (CPO) and cytochrome P-450 (P-450) have previously been used to suggest endogenous thiolate axial ligation to the CPO heme iron [1–6] as has been established for P-450. Both enzymes exhibit unique hyperporphyrin (‘split Soret’) spectra in their ferrous CO forms [1]. However, no free sulfhydryl group available for ligation to the heme iron have been found in chemical studies of CPO [7]. The t001. Soret Absorption Maxima (nm) and Spin States of the Ligand Complexes of Ferric CPO and P-450-CAM.aLigandCPObP-450-CAMcλ(ϵmM)Spin Statedλ(ϵmM)Spin StatedSH−449(e)ls467(43)flCH3COS−446(62)gls459elsCN−439(92)ls439(78)lsPyridine439(82)l421(111)lsNO437(114)ls430.5(105)lsCH3(CH2)3NC435(105)ls429.5(98)lsN−3432(110)ls427(81)lsSeCN−432(80)ls425(90)lsSCN−429(112)ls425(98)lsImidazole429(100)ls425(98)lsNo−2427.5(101)lseOCN−∼427els418.5(98)lsHCOO−425(121)ls419(106)lsHOCH2CH2SH418(69)ms464(58)fls417 (∼60)ms417(∼60)CH3CH2CH2SH416(74)ms464(∼33)flsCH3SCH3416(93)ms424(91)lsThioxane416(88)ms418(97)lsCH3SSCH3416(93)ms418(98)lsCH3COO−413(97)ms420(98)lsF−409(98)ghsno bindinga0.1 M K+ Phospate, 4 °C.bpH 6.0.cpH 7.0, (Refs.9–11).dls, low spin; ms, high-low mixed spin; hs, high spin.eUnstable.fRed Soret of hyperporphyrin spectrum (Ref. 11).gpH 3.0.t002. Soret Absorption Maxima (nm) of the Ligand Complexes of Ferrous CPO and P-450-CAM.LigandCPOa2 λ(ϵmM)P-450-CAMb2 λ(ϵmM)Me2PhPc2459d2460(98)CN−454(∼140)Not observedCH3(CH2)3NC452(141)452(106)CO444(167)446(120)NO440(114)438(83)a2pH 6.0.b2pH 7.0.c2Dimenthyllphenylphosphine.d2Not determined. MCD [4] and EPR [6] spectroscopic properties of analogous CPO and P-450 derivatives are generally similar, although some differences do exist. To obtain additional information about the active site structure of CPO, we have carried out extensive optical and MCD spectroscopic studies of both ferric and ferrous CPO in the presence of various exogenous ligands. The results obtained with CPO have been compared with those obtained with P-450 [9 - 11, this work] in order to obtain a better understanding of the electronic structure of the heme iron in both proteins.