Spectroscopic and computational studies on iron and manganese superoxide dismutases: nature of the chemical events associated with active-site pKs.
A combined spectroscopic/computational approach has been utilized to explore the chemical origins of the active-site pKs of the structurally homologous Fe- and Mn-dependent superoxide dismutases (SODs). Absorption, circular dichroism, magnetic circular dichroism, and variable-temperature, variable-field magnetic circular dichroism spectroscopic experiments have permitted us to determine electronic transition energies and polarizations, as well as ground-state spin Hamiltonian parameters. These experimental data have been used in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization configuration interaction (INDO/S-CI) computations for evaluating hypothetical active-site models for the high-pH species generated by density functional theory (DFT) geometry optimizations. Our experimental and computational data indicate that both reduced FeSOD and oxidized MnSOD do not bind hydroxide at high pH; rather, the active-site pK for these two species is attributed to deprotonation of a second-sphere tyrosine. Conversely, our data obtained on oxidized FeSOD indicate that hydroxide binding is responsible for the observed active-site pK for this species. Intriguingly, in the Fe-substituted form of MnSOD this identical chemical event occurs at a significantly lower pH. Overall, our results suggest an important role for second-sphere amino acids in tuning the active sites' interaction with small anions and bring into question the assumption that these homologous enzymes operate by the same molecular mechanism.
Jackson
Timothy A
TA
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
Xie
Juan
J
Yikilmaz
Emine
E
Miller
Anne-Frances
AF
Brunold
Thomas C
TC
D001665:Binding Sites; D002942:Circular Dichroism; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*
D005296:Ferrous Compounds; D008345:Manganese; D013482:Superoxide Dismutase