Ute Pfitzner

Cytochrome c Oxidase (Heme aa3) from Paracoccus denitrificans: Analysis of Mutations in Putative Proton Channels of Subunit I

One of the challenging features of energy-transducing terminal oxidases, like the aa3 cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen. As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis. The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochromec oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a α-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochromec yielded a kinetic difference spectrum resembling that of heme a 3. This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from CuA to the binuclear center may not occur via hemea but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Δψ generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

MUTATIONS IN THE CA2+ BINDING SITE OF THE PARACOCCUS DENITRIFICANS CYTOCHROME C OXIDASE

Recent structure determinations suggested a new binding site for a non‐redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site‐directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild‐type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp‐477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

Resonance Raman scattering from cytochromes aa3 and ba3 in the fully oxidized cytochrome oxidase /H2O2 reaction

Cytochrome oxidase, the terminal enzyme of the mitochondrial chain catalyzes the oxidation of reduced cytochrome c by molecular oxygen. Four different means have been used to produce and detect intermediate species in the reaction between dioxygen and quinol-or cytochrome -oxidizing terminal oxidase. Although the data to support the reaction sequence for the reduction of O2 by cytochrome oxidase are substantial, a troubling aspect of the work on the various intermediates has been the difficulty in establishing spectroscopic links between transient species in the O2 reduction observed during time-resolved measurements and metastable species generated by other techniques.