SERR-Spectroelectrochemical Study of a cbb₃ Oxygen Reductase in a Biomimetic Construct

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.

Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins

“…Spectra taken at sufficiently negative and positive potentials should be similar to spectra obtained in presence of a chemical reductant (dithionite) and oxidant (ferrocyanide), respectively. Such analyses were reported for cco in ptBLM assemblies Hrabakova et al , 2006 ;Todorovic et al , 2008 ) and confirmed that the structure of these proteins is largely preserved after immobilization.…”

“…These bands correspond to the C = O stretching and N-H in-plane bending vibrations, respectively, of the polypeptide chain (Barth , 2007 ). Within these lines, it was confirmed that the transmembrane helices of the cbb 3 oxidase adopt a near-normal orientation relative to the gold surface in protein-tethered BLM assemblies (Todorovic et al , 2008 ). Importantly the techniques allow the protein to be reconstituted in phospholipid membranes and thus allow us to study the effect of the membrane on the properties of the proteins.…”

“…Such mediated electron transfer electrocatalytic processes were reported for cco physisorbed on 3-mercapto-1-propanol modified gold electrodes (Haas et al , 2001 ) or for cco with a His-tag on subunit I in ptBLM assemblies (Friedrich et al , 2008 ). In the case of cbb 3 oxidase, an artificial redox mediator was used: N,N,N ′ ,N ′ -tetramethyl-p-phenylendiamine (Todorovic et al , 2008 ).…”

Immobilized Redox Proteins: Mimicking Basic Features of Physiological Membranes and Interfaces

“…A similar approach was applied to a fumarate reductase immobilized on Au electrode with hydrophobic coating (Kinnear and Monbouquette, 1993). An alternative immobilization method has been developed for proteins that contain a genetically introduced His tag Ataka et al, 2004;Giess et al, 2004;Hrabakova et al, 2006;Todorovic et al, 2008). After functionalizing the solid support with Ni (or Zn) NTA (3,3´-dithiobis[N-(5amino-5-carboxy-pentyl)propionamide-N, N´-diacetic acid)] dihydrochloride) monolayer, the protein can be attached via His coordination to the Ni center, Figure 3C.…”

“…It is purified from an organism that is capable of growing heterotrophically via fermentation and aerobic and anaerobic respiration, with a genetically introduced His-tag, allowing immobilization of the CcO on a metal electrode via Ni-NTA SAMs, Figure 3C Giess et al, 2004;Hrabakova et al, 2006;Todorovic et al, 2008). The protein was specifically attached, uniformly oriented and catalytically active in the biomimetic construct.…”