Optical detection of a red-shifted Michaelis complex in the reaction of HCN with ferromyeloperoxidase. Evidence for conformational stabilization of high spin iron (II).

Abstract

The reaction of HCN with ferromyeloperoxidase involves the sequential formation of two monocyanide complexes. The first complex, which forms immediately on mixing, is characterized by a red shift in the Soret band of the ferroperoxidase, and a dissociation constant (measured as a Michaelis constant) of 0.67 mM. The second complex arises from the first via a first order process, whose maximal rate is 0.095 s-1 at 25 degrees C, pH 7.0. This more stable complex is characterized by a blue shift in the Soret and alpha bands and by an overall dissociation constant in the region of 4.5 microM. This gives a free energy difference between the two complexes of around 3.0 kcal mol-1 and a difference in optical absorption of 15 nm (Soret). The measured Arrhenius activation energy for the conversion of the high energy, long wavelength complex to the low energy, short wavelength complex is 16.3 kcal mol-1. A larger blue shift is observed on protein denaturation (34 nm), after which the two-step binding reaction is not observed. This, and the magnitude of the activation energy in the spontaneous complex interconversion process, shows that the latter is a conformational process. In addition, it can be concluded that the unknown structural feature of the heme site which is responsible for the anomalous red shift in the optical spectrum of native ferromyeloperoxidase, is also the link between the ligand state of the iron and the protein conformation.