Temperature-Dependent Spin Equilibrium of Microsomal and Solubilized Cytochrome P-450 from Rat Liver

In the absence of exogenous substrate, temperature-induced type I and reverse type I (formerly called modified type II) spectral changes were observed with both rat liver microsomes and solubilized, partially purified cytochrome P-450. Similar changes were observed in the absolute spectrum of cytochrome P-450, i.e., increasing temperature reduced the absorption peak at 418 nm while simultaneously enhancing absorption in the 370-390-nm region in both liver preparations. An isosbestic point at 407 nm was noted and is indicative of a two-state equilibrium system. Spectral titration of type I and reverse type I spectral changes at 385 and 419 nm by dual wavelength spectrophotometry resulted in a slightly sigmoid-shaped curve with changes in temperature (3-40 °C). A graph of the log of the equilibrium constant vs. reciprocal temperature yielded a linear van't Hoff plot. From the slope and intercept an overall enthalpy change, ΔH = -14.66 kcal/mol, and entropy change, ΔS = -49.88 eu, in the reaction P-450LS P-450Hs were obtained for hepatic microsomes. These thermodynamic parameters yield an equilibrium constant of 0.930 at 20 °C, indicating a significantly large amount (>48%) of high-spin cytochrome P-450 in the absence of added substrate, as corroborated by electron paramagnetic resonance spectroscopy. In contrast, the soluble cytochrome P-450 preparation yielded ΔH = -5.5 kcal/mol and ΔS = -14.1 eu. These state functions produced an equilibrium constant of 0.096 at 20 °C indicative of P-450 predominantly in the low-spin ferric form. These differences suggest that the microsomal environment of lipid and/or endogenous substrate has a dramatic effect on controlling the spin equilibrium. In addition to these thermodynamic parameters, the precise absolute extinction coefficients for pure high-spin and pure low-spin cytochrome were derived. These parameters for solubilized P-450 of εHS = 52 mM-1 cm-1 and εLS = 126 mM-1 cm-1 at 418 nm and the corresponding values for the membrane-bound protein indicate that only 54% of microsomal P-450 undergoes a temperature-dependent spin transition. For the liver microsomes, Δε385_419nm was found to be 126 mM-1 cm-1.