Probing heart cytochromec oxidase structure and function by infrared spectroscopy

Abstract

IR spectra directly probe specific vibrators in bovine heart cytochrome c oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to Fe2+ a3 as two conformers each in isolated immobile environments sensitive to Fea and/or CuA oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN- that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fea, CuA, and CuB with associated changes in protein conformation of likely significance in O2 reduction and H(+)-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of alpha-helix (approximately 60%) and subtle changes with redox level and exposure to anesthetics. N2O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.

Anesthetic-like Interactions of Nitric Oxide with Albumin and Hemeproteins

“…In each case, curve-fitting revealed one fitted band that was consistent with a N-O stretching band with parameters identical to those found earlier when the reference solution used contained 15 for HbNO) (19,28). The problems associated with isolating the true N-O stretching band from the protein Amide I bands that appear in the same region of the IR spectrum have been discussed (10,19,28). Effects of N 2 -flushing on the spectrum can be attributed to changes in protein secondary structure as the result of the removal of NO from noniron sites from which NO dissociates more rapidly than from the "slow release" sites discussed above.…”

“…The absorbance recorded represents the sum of the true N-O stretching vibration plus the difference between the Amide I spectrum of the sample solution and the Amide I spectrum of the reference solution when a CO-saturated solution of the respective protein carbonyl was used as reference. In each case, curve-fitting revealed one fitted band that was consistent with a N-O stretching band with parameters identical to those found earlier when the reference solution used contained 15 for HbNO) (19,28). The problems associated with isolating the true N-O stretching band from the protein Amide I bands that appear in the same region of the IR spectrum have been discussed (10,19,28).…”

“…Both NO and NNO inhibit CcO reversibly (18,48). Since NNO does not serve as a ligand to these metals, the partial, reversible inhibition by NNO appears due to the noncovalent bonding of NNO molecules to one or more of the nonmetal sites that are detected in IR spectra (5,18,28). Noncovalent bonding of NO to NNO sites, and possibly other sites, provides new mechanisms for the control of CcO activity by NO.…”

“…Hemeprotein Studies-Hemeprotein solutions were prepared in 200 mM sodium phosphate buffer, pH 7.2, under strictly anaerobic conditions as described earlier (19,28). Several methods were used to detect the relative affinities of the proteins for NO and CO and the slow release of NO.…”

IR signatures of the metal centres of bovine cytochrome c oxidase: assignments and redox-linkage

“…CO ligation was achieved with CO saturated redox buffer and a reducing potential of − 400 mV; the binding extent was monitored from the intensity of the FR-CO (fully reduced CO-ligated) band at 1963 cm − 1 . CN ligation was achieved by inclusion of 10 mM KCN in the redox buffer and an oxidizing potential of + 500 mV; binding was monitored from the intensity of the O-CN (fully oxidized cyanideligated) band at 2151 cm − 1 [41].…”