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

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.a Ligand CPOb P-450-CAMc λ(ϵmM) Spin Stated λ(ϵmM) Spin Stated SH− 449(e) ls 467(43)f l CH3COS− 446(62)g ls 459e ls CN− 439(92) ls 439(78) ls Pyridine 439(82) l 421(111) ls NO 437(114) ls 430.5(105) ls CH3(CH2)3NC 435(105) ls 429.5(98) ls N−3 432(110) ls 427(81) ls SeCN− 432(80) ls 425(90) ls SCN− 429(112) ls 425(98) ls Imidazole 429(100) ls 425(98) ls No−2 427.5(101) ls e OCN− ∼427e ls 418.5(98) ls HCOO− 425(121) ls 419(106) ls HOCH2CH2SH 418(69) ms 464(58)f ls 417 (∼60) ms 417(∼60) CH3CH2CH2SH 416(74) ms 464(∼33)f ls CH3SCH3 416(93) ms 424(91) ls Thioxane 416(88) ms 418(97) ls CH3SSCH3 416(93) ms 418(98) ls CH3COO− 413(97) ms 420(98) ls F− 409(98)g hs no binding a 0.1 M K+ Phospate, 4 °C. b pH 6.0. c pH 7.0, (Refs.9–11). d ls, low spin; ms, high-low mixed spin; hs, high spin. e Unstable. f Red Soret of hyperporphyrin spectrum (Ref. 11). g pH 3.0. Full-size table Table options View in workspace Download as CSV t002. Soret Absorption Maxima (nm) of the Ligand Complexes of Ferrous CPO and P-450-CAM. Ligand CPOa2 λ(ϵmM) P-450-CAMb2 λ(ϵmM) Me2PhPc2 459d2 460(98) CN− 454(∼140) Not observed CH3(CH2)3NC 452(141) 452(106) CO 444(167) 446(120) NO 440(114) 438(83) a2 pH 6.0. b2 pH 7.0. c2 Dimenthyllphenylphosphine. d2 Not determined. Full-size table Table options View in workspace Download as CSV 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. CPO was isolated and purified (A399/A280 > 1.4) from C. fumago as reported previously [8]. The purification of P-450-CAM and the procedure and instrumentation used for the ligand binding experiments and spectroscopic measurements have been previously reported [9–11]. A wide variety of exogenous anionic and neutral ligands, including several not previously known to bind to ferric or ferrous CPO, form complexes with the enzyme having Kd values ranging from 10−5 to 10−1M (pH 3–6, 4°C). The Soret region optical absorption coefficients of the ferric and ferrous CPO and P-450 derivatives are summarized in Tables I and II, respectively. For ferric CPO, the absorption maxima vary from 409–449 nm. A similar range is found for the analogous ferric P-450-CAM adducts (Table I). All ferrous CPO and P-450 ligand complexes have Soret maxima in a narrow range from 438–460 nm (Table II). Addition of a strongly acidic (pKa ∼ 3.5) thiol, thioacetic acid, to ferric CPO yields a complex that has a hyperporphyrin absorption spectrum that is very similar to that of the ferric P-450-CAM·benzylmercaptan adduct (Fig. 1), a well characterized bisthiolate heme complex [11]. Furthermore, MCD spectrum of the ferric CPO·thioacetic acid adduct also exhibits a band pattern that is typical of hyperporphyrin complexes [11]. Together, these data suggest that the CPO—thiolate complex has a bisthiolate heme coordination structure; clearly, such a deduction requires an endogenous thiolate ligand in the native enzyme. The UV-visible absorption and MCD spectra of ferrous CPO and P-450-CAM adducts with isocyanides are presented in Fig. 2. While very similar overall, some spectral differences are observed, especially in the more intense visible region MCD features of the CPO adduct. The MCD spectra of the ferrous NO and ferric KCN derivatives of CPO and P-450-CAM are displayed in Fig. 3. As has been observed for native ferric, ferrous and ferrousCO CPO and P-450-CAM [4], the MCD spectral features of the NO or KCN adducts of both enzymes are similar except for some minor intensity and line shape differences. In contrast to the above spectral similarities observed between analogous CPO and P-450-CAM derivatives, we have observed significant differences between the two enzymes in the spin state distribution of certain ferric ligand adducts. Although most anionic ligands listed in Table I and some neutral ligands (pyridine, isocyanide, imidazole and NO) from exclusively low spin complexes with ferric CPO, the adducts with other ligands such as neutral sulfur donors are instead a mixture of high and low spin. In addition, formate (low spin) and acetate (mixed spin) behave differently even though both are carboxylate anions at pH 6. Finally, among the heme ligands studied, only fluoride forms a high spin complex with ferric CPO. These diverse spin state properties of the ferric CPO ligand complexes stand in contrast to the uniformly low spin nature of all ferric P-450-CAM ligand complexes [10]. In conclusion, the hyperporphyrin spectra that are observed for the complex obtained through ligation of a strongly acidic thiol to ferric CPO provide compelling evidence for the presence of an endogenous thiolate ligand to the heme iron of the ferric enzyme. The additional spectral similarities observed between analogous ligand complexes of CPO and P-450-CAM add further support to this conclusion for both the ferric and ferrous CPO cases. However, since ligand complexes of P-450-CAM and thiolate-ligated heme models are exclusively low spin, the spin state distribution distinctions seen between some weak field ligand complexes of ferric CPO and P-450 suggests that the heme-iron: thiolate-sulfur interaction in chloroperoxidase is somewhat different from that in P-450 and model complexes. Additional work is in progress to try to identify the cause of these differences.