No abstract

The cbb3 oxygen reductase from Bradyrhizobium japonicum was immobilized on nanostructured silver electrodes by anchoring the enzyme via a His-tag to a Ni-NTA coating, followed by reconstitution of a lipid bilayer. The immobilized enzyme retains the native structure and catalytic activity as judged by in situ surface-enhanced vibrational spectroscopy and cyclic voltammetry, respectively. Spectroelectrochemical titrations followed by SERR spectroscopy of the integral enzyme and its monohemic (fixO) and dihemic subunits (fixP), allowed the determination of the reduction potentials for the different heme c groups. Both in the isolated subunits and in the integral enzyme the Met/His-coordinated hemes from the two subunits present identical reduction potentials of 180 mV, whereas for the bis-His heme from fixP the value is ca. 400 mV. The determination of reduction potentials of the individual hemes c reported in this work provides the basis for further exploring the mechanism of electroprotonic energy transduction of this complex enzyme.

Haem-copper oxygen reductases are transmembrane protein complexes that reduce dioxygen to water and pump protons across the mitochondrial or periplasmatic membrane, contributing to the transmembrane difference of electrochemical potential. Seven years ago we proposed a classification of these enzymes into three different families (A, B and C), based on the amino acid residues of their proton channels and amino acid sequence comparison, later supported by the so far identified characteristics of the catalytic centre of members from each family. The three families have in common the same general structural fold of the catalytic subunit, which contains the same or analogous prosthetic groups, and proton channels. These observations raise the hypothesis that the mechanisms for dioxygen reduction, proton pumping and the coupling of the two processes may be the same for all these enzymes. Under this hypothesis, they should be performed and controlled by the same or equivalent elements/events, and the identification of retained elements in all families will reveal their importance and may prompt the definition of the enzyme operating mode. Thus, we believe that the search for a minimum common denominator has a crucial importance, and in this article we highlight what is already established for the haem-copper oxygen reductases and emphasize the main questions still unanswered in a comprehensive basis.