CHARACTERIZATION OF IRON-SULFUR CLUSTERS IN LYSINE 2,3-AMINOMUTASE BY ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion Of L-a-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 muW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which g(xx) = 2.027, g(yy) = 2.007, and g(zz) = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S]. The signal of the [3Fe-4S] cluster undergoes power saturation at 10 K in a homogeneous fashion, in which p1/2 = 10 +/- 3 mW. The EPR spectrum of the [3Fe-4S] cluster is simulated by a rhombic spectrum, in which g(xx) = 2.032, g(yy) = 2.015, and g(zz) = 2.0125. Double integration of the [3Fe-4S] spectrum indicates the presence of 2.5-3 spins per hexameric unit of enzyme. Reductive activation of the enzyme leads to the disappearance of the EPR signal of the [4Fe-4S] cluster, at a rate that is significantly faster than the activation process, and to the quenching of the radical signal at a rate that is comparable to the rate of activation. The reduction potential of the [4Fe-4S] cluster is more positive than -370 mV.