title: 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.
abstract: Cytochrome c oxidase (CcO), the terminal oxidase of cellular respiration, reduces molecular oxygen to water. The mechanism of proton pumping as well as the coupling of proton and electron transfer is still not understood in this redox-linked proton pump. Eleven residues at the aqueous-exposed surfaces of CcO from Paracoccus denitrificans have been exchanged to cysteines in a two-subunit base variant to yield single reactive cysteine variants. These variants are designed to provide unique labeling sites for probes to be used in spectroscopic experiments investigating the mechanism of proton pumping in CcO. To this end we have shown that all cysteine variants are enzymatically active. Cysteine positions at the negative (N-) side of the membrane are located close to the entrance of the D- and K-proton transfer pathways that connect the N-side with the catalytic oxygen reduction site. Labeling of the pH-indicator dye fluorescein to these sites allowed us to determine the surface potential at the cytoplasmic CcO surface, which corresponds to a surface charge density of -0.5 elementary charge/1000Å(2). In addition, acid-base titrations revealed values of CcO buffer capacity. Polarity measurements of the label environment at the N-side provided (i) site-specific values indicative of a hydrophilic and a more hydrophobic environment dependent on the label position, and (ii) information on a global change to a more apolar environment upon reduction of the enzyme. Thus, the redox state of the copper and heme centers inside the hydrophobic interior of CcO affect the properties at the cytoplasmic surface.
authors.affiliation: Physics Department, Freie Universität Berlin, Berlin, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kirchberg
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
authors.forename: Ulrike
authors.initials: U
authors.lastname: Alexiev
mesh_terms: D002021:Buffers; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons
chemical_list: D002021:Buffers; D011522:Protons; D003576:Electron Transport Complex IV