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.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
Timothy A
TA
Jackson
Juan
J
Xie
Emine
E
Yikilmaz
Anne-Frances
AF
Miller
Thomas C
TC
Brunold
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