Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy.

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.