Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues

We re-determined the near infrared (NIR) spectral signatures (650–980 nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980 nm is cupric Cu A , which in the beef heart enzyme has a maximum at 835 nm. The 655 nm band characterises the fully oxidised haem a 3 /Cu B binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting ‘perturbed’ binuclear centre is also characterised by a previously unreported broad 715–920 nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835 nm maximum or the 715 nm band. Our results confirm the ability of NIRS to monitor the Cu A centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a 3 /Cu B binuclear centre. • The NIR spectrum of cytochrome oxidase was deconvoluted into its component species. • The dominant feature between 700 and 980 nm was confirmed as the CuA chromophore. • There was no significant contribution from the haem a iron centre. • A new feature between 715 and 920 nm was assigned to the haem a 3 /Cu B binuclear centre. • Changes in concentrations of oxygen intermediates P and F may be measurable in vivo.