Electrochemical and FTIR Spectroscopic Characterization of the Cytochrome bc₁ Complex from Paracoccus denitrificans:  Evidence for Protonation Reactions Coupled to Quinone Binding

Abstract

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.

Differences in Protonation of Ubiquinone and Menaquinone in Fumarate Reductase from Escherichia coli

“…Redox-induced FTIR Difference Spectra-The vibrational modes of quinones in their different redox and protonation states can serve as a reporter group for steric and energetic factors such as hydrogen bonding, polar interactions, and distortion of the ring and constituents. This has been previously demonstrated for several systems, including the light-induced bacterial reaction center (27,28), electrochemically induced bc 1 complex (29,30), E. coli cytochromes bo (31) and bd (32), and QFR from Wolinella succinogenes (33). Fig.…”

“…Additionally, proton reactions concomitant with electron transfer are expected to contribute in the spectra. The data display a major differential feature between 1700 and 1620 cm Ϫ1 , as found typically for iron-sulfur proteins (24,30). These signals in the so-called amide I range involve the (CϭO) stretching vibration of the polypeptide backbone reorganizing upon electron transfer in the cluster.…”

“…6, the split (CϭO) modes of the oxidized quinone are at 1652 cm Ϫ1 with a shoulder at 1664 cm Ϫ1 and the (CϭC) mode is at 1611 cm Ϫ1 . At 1288 and at 1264 cm Ϫ1 the signals from the C-OCH 3 vibrations of the 2-and 3-methoxy groups contribute (27)(28)(29)(30)(31)(32)37 . These two bands are very similar to those from ubiquinone in solution, and they have been found to be largely insensitive to the protein environment (27)(28)(29)(30)(31).…”

Asymmetric Binding of Stigmatellin to the Dimeric Paracoccus denitrificans bc1 Complex

“…The Y159F mutant Rieske protein was assumed to have a E m of 221 mV based on a ⌬E m of Ϫ44 mV with respect to the 265-mV value reported for the wild type (13,21). The lowest in the range of E m values reported for the P. denitrificans cytochrome c 1 (22) was used (268 mV). E m values for heme b H and b L were assumed to be ϩ58 and Ϫ92 mV, respectively (22).…”

“…The lowest in the range of E m values reported for the P. denitrificans cytochrome c 1 (22) was used (268 mV). E m values for heme b H and b L were assumed to be ϩ58 and Ϫ92 mV, respectively (22). Using the Nernst equation, the ⌬E m of 79 mV between DBH 2 and the average E m for the Rieske protein and the b H heme (139.5 mV) was used to set a forward/reverse rate ratio of 4.7 for the first turnover at center P. For the second turnover, an average E m of 64.5 mV for the Rieske protein and the b L heme was used to set a rates ratio of 1.08.…”