The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium

Abstract

We have investigated the spin-state equilibrium of adrenal mitochondrial P450,,, (cholesterolside-chain-cleaving, CYPl1 Al) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 "C to -20 "C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are AH, = 0.7 kJ . mol-' andat low temperatures and AH, = -42 kJ.mol-' and AS, = -152 J . K-' . mol-' at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH.The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.The cytochrome P450 (P450) heme-iron spin state has intrigued researchers for many years [l]. Upon substrate binding, P450 converts in most cases from a low-spin ( S = 1/2) to a high-spin ( S = 5/2) state. The spin state is coupled to the P450 redox potential: P450 reduction is more easy in the highspin than in the low-spin state [2,3], suggesting a link between spin state and enzyme function. Both, substrate-free and substrate-bound P450 are reported to exist in a thermal spin-state equilibrium [ l , 2, 41. In fact, most spin investigations are indirect : they deal with the spectrally determined equilibrium between P450 species absorbing at 417 nm and 391 nm. However, the position of the absorbance maximum in the Soret band of the ferric protein is related to the spin state of its iron center, as measured by resonance techniques such as EPR and Mossbauer spectroscopy [2, 4, 51. During a low-spin to highspin transition, the heme iron looses its sixth ligand and becomes displaced 45 pm out of the heme plane [6]. This heme structural change depends however on the ligand field produced by the surrounding protein and by the bound substrate. It is influenced by a wide spectrum of physiological and Abbreviations. P450,,,, cholesterol-side-chain-cleaving cytochrome P450; P450,,,, camphor monooxygenase.Enzymes. Cytochrome P450,,,, cholesterol monooxygenase (sidechain-cleaving) (EC 1.14.15.6); cytochrome P450,,,, camphor monooxygenase (EC 1.14.15.1).Mende (CNRS), F-34033 Montpellier, France physicochemical parameters [7]. The spin state is therefore a potentially helpful parameter for the detection of proteinconformational changes. Especially, the thermodynamic parameters AH and AS of this reaction have been used to unravel discrete protein-conformational changes [4, 8, 91. Whereas the spin-state thermodynamics of ba...

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

“…Almost complete conversion of cholesteroldepleted hemoprotein preparations to the high-spin state [15,163,164] was attained upon tight complexation with exogenously added substrate [165]; this transition was mirrored in an increase in the distance of water protons from the heme iron as a manifestation of the displacement of the aqua ligand [166]. Spin equilibria were found to be sensitive to pH and temperature changes [167]. In addition, intermediates and final product of the cholesterol desmolase reaction turned out to bind CYP11A1 very tightly [162], however, the effectiveness at inducing a high-spin shift strongly differed between the various compounds: While 20R,22R-dihydrocholesterol yielded a completely high-spin cytochrome, 22R-hydrocholesterol and pregnenolone behaved anomalously in producing entirely low-spin enzyme [15,162]; the latter conformer might have originated from contribution by the oxygenated derivatives of a sixth hydroxy ligand interacting with the heme iron in a disordered mode [168] ( Table 2).…”

“…Finally, attention has to be drawn to the experimental setup designed to assess the impact of substrate on spin equilibria. As is known, spin transition in P450s is highly prone to thermal effects [32,43,72,129,167], so that proper conclusions as to spin-state changes at physiological temperatures might be impeded by the use of information from Mössbauer [12] and EPR [11,16,98,116,157,179] spectral measurements carried out under cryogenic conditions. However, this possibility seems to be of minor importance, since careful examination revealed more than 80% of the spectral data discussed in the present review to originate from analyses by UV-visible absorption spectroscopy performed at ambient temperatures.…”

Activation volume and energetic properties of the binding of CO to hemoproteins

“…In this study we have paid a particular attention to cytochrome P-450SCC. In a previous thermodynamic investigation, we had characterized the dynamics and the nature of conformational changes which occur as a result of changes in pH and temperature (Lange et al, 1992(Lange et al, , 1992a). This study revealed that the latter parameters had no effect on the activation volume of the binding rate constant.…”

Solution behavior of iron(iii) and iron(ii) porphyrins in DMSO and reaction with superoxide. Effect of neighboring positive charge on thermodynamics, kinetics and nature of iron-(su)peroxo product

“…All chemicals were used as received without further purification. K 18 O 2 was prepared according to a published method; 6 18 O 2 was obtained from Aldrich.…”

“…The sample of the starting complex [Fe III (tBuTPP)Cl] was prepared by making a 5 ¥ 10 -4 M suspension in dry DMSO (stirred overnight) under nitrogen and subsequent filtration. Other samples were prepared by dissolving excess of either KO 2 or K 18 O 2 in an overnightprepared 5 ¥ 10 -4 M suspension of [Fe III (tBuTPP)Cl] in dry DMSO under nitrogen and subsequent filtration. High mass accuracy ESI spectra were recorded on an ultra high-resolution ESI-Time-Of-Flight MS, a Bruker Daltoniks (Bremen, Germany) Maxis, which also was coupled to the Bruker cryospray.…”