The effect of cytochrome P-450cam on the NMR relaxation rate of water protons.
J Biol Chem, 1979/10/25;254(20):10173-9.
Philson SB, Debrunner PG, Schmidt PG, Gunsalus IC
PMID: 226536
Abstract
Cytochrome P-450cam in the native, substrate-free state (Fe3+, S = 1/2) substantially reduces the NMR relaxation times, T1 and T2, of water protons. Temperature and frequency dependences of T1 and T2 were measured; they are consistent with a model of one or two protons exchanging between a binding site on a heme ligand and bulk water. The relevant parameters of this model have been deduced from the data. The spin relaxation time of the heme iron, tau S similar to 0.5 ns at 25 degrees C, is unusually long for a low spin ferric heme protein but is compatible with the line widths measured for paramagnetically shifted heme resonances. The proton residence time on the ligand, tau M similar to 1 microsecond at 25 degrees C, follows an Arrhenius law with activation energy EM similar to 15 kcal/mol. A scalar hyperfine interaction A/h = 2.2 MHz (3.1 MHz for one-proton exchange) of the found proton(s) with the heme iron is deduced from the difference between T1 and T2 observed in the fast exchange limit. The iron-proton distance is found to be 2.9 A (2.6 A for one-proton exchange). Variation of pH between pH 6.4 and 8.6 does not affect T1. The bearing of these results on the question of the axial heme ligand is discussed.
MeSH terms
Binding Sites; Cytochrome P-450 Enzyme System; Electron Spin Resonance Spectroscopy; Heme; Iron; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Protein Binding; Protein Conformation; Protons; Temperature; Water
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