ENDOR studies of pyruvate: ferredoxin oxidoreductase reaction intermediates

Electron−nuclear double resonance (ENDOR) studies of radical intermediates formed by the oxidative decarboxylation of pyruvate by pyruvate:ferredoxin oxidoreductase were carried out to characterize their electronic structure and elucidate aspects of the recently proposed catalytic mechanism (Menon, S.; Ragsdale, S. W. Biochemistry 1997, 36, 8484−8494). The EPR spectrum of the PFOR/pyruvate adduct at 4 K displays a narrow resonance centered at g = 2.008 that has been attributed to a hydroxyethyl thiamine pyrophosphate (HE-TPP) radical. This spectral feature is superimposed on a broad, complex line shape characteristic of magnetically coupled [Fe4S4] clusters. The ENDOR spectrum at g = 2.008 reveals a broad peak with a complex line shape that can be analyzed, assuming that it arises from a composite of two axially symmetric proton hyperfine couplings. The principle coupling values for these two hyperfine tensors were:  A∥(1) = 18.9 MHz, A⊥(1) = 12.6 MHz; and A∥(2) = 20.3 MHz, A⊥(2) = 14.9 MHz. The assignmen...