CLUSTER STRUCTURE AND H-BONDING IN NATIVE, SUBSTRATE-BOUND, AND 3FE FORMS OF ACONITASE AS DETERMINED BY RESONANCE RAMAN-SPECTROSCOPY

Resonance Raman spectra, with excitation in the visible and near-UV regions, have been investigated for aconitase with and without substrate and inhibitors, using S-34, O-18, and H-2 labeling of the active site or substrate. The Fe-O stretching vibrations of bound hydroxide, substrates, or inhibitors are not resonance enhanced. However, their influence is detectable in O-18 shifts of FeS modes of the [Fe4S4b]S-3(t) cluster and in the frequency elevation of one of the FeSt modes from ca. 360 to 372 cm(-1). The FeS modes have all been assigned. Their frequencies and S-34(b) shifts are reproduced via normal mode calculations on a cluster model with ethyl thiolate ligands, provided that the FeS-CC dihedral angles are set to the values found in the crystal structures and that allowance is made for FeS force constant differences due to weakening of the bonds to the unique Fe atom, Fe-a, to which OH is attached and strengthening of the bonds to the remaining Fe atoms. The frequencies and isotope shifts are likewise, reproduced for the 3Fe inactive form of aconitase if allowance is made for further strengthening of the bonds to the doubly bridging S atoms, once Fe-a is removed. D2O shifts of 2 cm(-1) are seen for the E symmetry FeSb cluster mode and for an FeSt mode at 358 cm(-1) for aconitase with bound substrate, consistent with the H-bonds to both bridging and terminal S atoms deduced from the crystal structure. In native aconitase, the 360-cm(-1) FeSt band likewise shifts 2 cm(-1) in D2O, and the intensification of this band upon substrate or inhibitor binding indicates an alteration in the excited state of the H-bond interaction with a terminal sulfur atom. In addition, resonance enhancement is observed for amide modes involving C=O stretching (amide I, 1655 cm(-1)) and C-C-N bending (466 cm(-1)), identified via their D2O sensitivity. The 1655-cm(-1) amide I band shifts to 1642 cm(-1) when aconitase is dissolved in D2O, but to 1624 cm(-1) when it is reconstituted from apoprotein in D2O. These shifts are consistent with H/D exchange of one and then both protons on a primary amide. It is concluded that the amide modes arise from one or both of the asparagine side chains involved in the H-bonds and that their enhancement arises from electronic coupling with the resonant Fe --> S charge-transfer transition via the H-bonds to the cluster.