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 re gions, 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, subs trates, or inhibitors are not resonance enhanced. However, their influ ence is detectable in O-18 shifts of FeS modes of the [Fe4S4b]S-3(t) c luster 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 frequ encies 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 structur es and that allowance is made for FeS force constant differences due t o weakening of the bonds to the unique Fe atom, Fe-a, to which OH is a ttached and strengthening of the bonds to the remaining Fe atoms. The frequencies and isotope shifts are likewise, reproduced for the 3Fe in active form of aconitase if allowance is made for further strengthenin g of the bonds to the doubly bridging S atoms, once Fe-a is removed. D 2O shifts of 2 cm(-1) are seen for the E symmetry FeSb cluster mode an d for an FeSt mode at 358 cm(-1) for aconitase with bound substrate, c onsistent with the H-bonds to both bridging and terminal S atoms deduc ed from the crystal structure. In native aconitase, the 360-cm(-1) FeS t band likewise shifts 2 cm(-1) in D2O, and the intensification of thi s band upon substrate or inhibitor binding indicates an alteration in the excited state of the H-bond interaction with a terminal sulfur ato m. In addition, resonance enhancement is observed for amide modes invo lving 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 ba nd shifts to 1642 cm(-1) when aconitase is dissolved in D2O, but to 16 24 cm(-1) when it is reconstituted from apoprotein in D2O. These shift s are consistent with H/D exchange of one and then both protons on a p rimary amide. It is concluded that the amide modes arise from one or b oth of the asparagine side chains involved in the H-bonds and that the ir enhancement arises from electronic coupling with the resonant Fe -- > S charge-transfer transition via the H-bonds to the cluster.