Temperature-Dependent Coordination in E. coli Manganese Superoxide Dismutase

Abstract

Two different temperature dependences of the manganese(II) high-field electron paramagnetic resonance spectrum of manganese superoxide dismutase from E. coli were observed. In the 25-200 K range, the zero-field interaction steadily decreased with increasing temperature. This was likely due to the thermal expansion of the protein. From these results, it was possible to deduce an approximately r(-)(2.5) dependence of Mn(II) zero-field interaction on ligand-metal distance. At temperatures above 240 K, a distinct six-line component was detected, the amplitude of which decreased with increasing temperature. On the basis of similarities to the six-line spectrum observed for the azide-complexed E. coli manganese superoxide dismutase, the newly detected six-line spectrum was assigned to a hexacoordinate Mn(II) center resulting from the coordination of a nearby water molecule to the normally five-coordinate center. The changes in enthalpy and entropy characterizing the hexacoordinate-pentacoordinate equilibrium in the 240-268 K range were -5 kcal/mol and -24 cal/mol.K, respectively. The structural implications of the zero-field parameters of the newly found hexacoordinate form in comparison to those of the Mn(II) centers in concanavalin-A and manganese-containing R. spheroides photosynthetic reaction centers and the values predicted by the superposition model are discussed.

Role of Tyrosine-34 in the Anion Binding Equilibria in Manganese(II) Superoxide Dismutases

“…The incubation of E. coli Mn(II)SOD at temperatures between 240 and 273 K prior to flash-freezing in liquid nitrogen has been shown to lead to the appearance of a six-line component in the middle of the HFEPR spectra, the magnitude of which was temperature dependent. This demonstrated that there was a temperaturedependent equilibrium between hexa and pentacoordinated centers (40). The same phenomenon was observed for both wild-type and mutant RcMn(II)SOD proteins (Figure 4).…”

“…A simulation of the spectra showed that the |D| and E zero-field parameters were 10.775 and 0.508 GHz, respectively, in RcMn(II)SOD-WT and 10.777 and 0.460 GHz, respectively, for the Y34F mutant ( Figure 3B). In both cases, as before with other Mn(II)SODs (21,40), distributions of 0.100-0.140 GHz in the |D| and E parameters, respectively, were required to obtain good fits. Although the differences between the wild-type and mutant zero-field parameters were within the estimated error of (0.06 GHz of the fitting procedure, the shift of 0.048 GHz in the E values appeared to be significant.…”

“…Because this transition occurred in absence of any exogenous ligand, we attributed this to the movement of a water molecule into the proximal state (40). In the resting state of the protein at room temperature, a water molecule lies in the distal state, as is seen in crystal structures of native MnSODs (44,45) and also the Y34F mutants (27,28).…”

“…For a given temperature, the normalized intensity of the six-line component was bigger for the RcMn(II)SOD-Y34F than for the wild-type enzyme. Each spectrum was then decomposed into two components, in the same manner as that we had previously done for the E. coli Mn(II)SOD (40). From the resulting data, the equilibrium constant, defined as the ratio of the hexacoordinated to pentacoordinated forms, at each temperature was estimated, and a van't Hoff plot was constructed ( Figure 5).…”

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…The EPR at multiple frequencies (MF EPR) approach is well illustrated by recent high-field studies that precisely determined ZFS parameters for MnSODs from a variety of organisms [42,63,64]. Both Mn-and Fe-containing SODs possess nearly identical first coordination spheres, yet Fe-substituted MnSOD and Mn-substituted FeSOD (Mn-(Fe)SOD) are both inactive.…”