Electrochemical and FTIR spectroscopic characterization of the cytochrome bc1 complex from Paracoccus denitrificans: evidence for protonation reactions coupled to quinone binding.
The cytochrome bc(1) complex from Paracoccus denitrificans and soluble fragments of its cytochrome c(1) and Rieske ISP subunits are characterized by a combined approach of protein electrochemistry and FTIR difference spectroscopy. The FTIR difference spectra provide information about alterations in the protein upon redox reactions: signals from the polypeptide backbone, from the cofactors, and from amino acid side chains. We describe typical modes for conformational changes in the polypeptide and contributions of different secondary structure elements. Signals attributed to the different cofactors can be presented on the basis of selected potential steps. Modes associated with bound quinone are identified by comparison with spectra of quinone in solution at 1656, 1642, and 1610 cm(-1) and between 1494 and 1388 cm(-1), as well as at 1288 and 1262 cm(-1). Signals originating from the quinone bound at the Q(o) site can be distinguished. On the basis of the infrared data, the total quinone concentration is determined to be 2.6-3.3 quinones per monomer, depending on preparation conditions. The balance of evidence supports the double-occupancy model. Interestingly, the amplitude of the band at 1746 cm(-1) increases with quinone content, reflecting a protonation reaction of acidic groups. In this context, the involvement of glutamates and/or aspartates in the vicinity of the Q(o) site is discussed on the basis of recently determined crystal structures.
Ritter
Michaela
M
Institut für Biophysik, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, Haus 74, 60590 Frankfurt am Main, Germany.
Anderka
Oliver
O
Ludwig
Bernd
B
Mäntele
Werner
W
Hellwig
Petra
P
D016227:Benzoquinones / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods*; D014450:Electron Transport Complex III / Q000737:chemistry / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D016227:Benzoquinones; D011522:Protons; C004532:quinone; D014450:Electron Transport Complex III