Exploring the entrance of proton pathways in cytochrome c oxidase from Paracoccus denitrificans: Surface charge, buffer capacity and redox-dependent polarity changes at the internal surface

► Eleven single-reactive cysteine variants of cytochrome c oxidase (CcO) from P. denitrificans were constructed. ► Modification of the cysteine residues with the fluorescent dye fluorescein was demonstrated. ► A distinct polarity pattern for the fluorescein attachment sites at the entrance of the D- and K-channel of CcO was observed. ► The cytoplasmic surface of CcO becomes more apolar upon reduction of the enzyme.