Electron spin resonance study of single crystals of cytochrome P450 from Pseudomonas putida
The magnetic properties of the ferric heme iron in single crystals of cytochrome P450 have been studied by electron spin resonance. Spectra were recorded for orthorhombic crystals (space group P222l , 114 molecules/ unit cell) of the native, substrate-free enzyme (mo),which showed a single signal of spin S=1/2, and of the camphor complex (mos), which showed a mixture of high-spin, S=5/2, and low-spin, 8=1/2, species. Complete angular scans were taken at 9.18 GHz and temperatures of 5K' (high-spin) or 15K (low-spin) to determine the orientation of the g-tensors with respect to the crystal axes. The results are expressed in terms of the Euler angles (alpha, beta, gamma) rotating the crystal axes (c,b,a) onto the g-tensor axes (x,y,z). The O best fits of (alpha, beta, gamma) were (0,-80.-100) for m , (3,-70,-103) for 8=1/2 os os m , and (O,-95.~115) for 8=5/2 m , ,where an accidental symmetry produces the sign ambiguity. The g-values of the various species were found to be (2.48,2.25, 1.91) for mO. (2.40,2.23.1.97) for 8=1/2 mOs, and (7.75,3.93,1.80) for S=5/2 mOs; these all differed slightly from the frozen solution values. The low-spin mossignals were purely from the 1.97 species as opposed to the mixture of 1.97 and 1.91 heretofore observed in solutions. The low-spin g=2.4 was assigned to g on the basis of its near coincidence with z the high-spin g=1.8 direction. which is known to be the heme normal. The linewidths measured in the single crystal studies were consistent with the crystal mosaic distortion plus g-strain model. Best fits for the linewidth simulations were obtained with the parameter values: delta-theta = 2.2Sofor crystal mosaic spread, delta-Delta = O.ISA, deltaV = O.ISA for low-spin g-strain, and delta-alpha = 0.004 for high-spin g-strain. Unresolved hyperfine broadening was assumed to be ISG in both species based on earlier heme protein studies.
electron spin resonance, single crystals, cytochrome P450, Pseudomonas putida, ferric heme iron
University of Illinois - Urbana-Champaign
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