The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy

Abstract

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri)

“…In our model the centre labelled A is shown to be clearly similar in structure to Cu, in bovine cytochrome c oxidase [15,16]. Although the form of A is controversial, the 7-line hyperfine structure in the EPR spectrum of this centre has found an explanation so far only in terms of a binuclear mixed-valence metal dimer 161.…”

Exploring the proton pump mechanism of cytochrome c oxidase in real time

“…2 A) has a spectrum distinct from that of Cu A in the two earlier phases (data not shown), and must therefore be ascribed to other processes (see below). It has indeed been demonstrated that the near-infrared absorbance at 820 nm cannot be completely ascribed to Cu A (27).…”

Spectroscopic and Mutagenesis Studies on the CuA Centre from The Cytochrome‐c Oxidase Complex of Paracoccus Denitrificans

“…3 of the MCD spectra at 4.2 K of the R denitr$icans Cu, domain, the Cu, centre deconvoluted from the MCD spectrum of bovine heart cytochrome-c oxidase (Greenwood et al, 1983), and that of the mutant form of nitrous-oxide reductase (N,ORV). The spectrum of Cu, in bovine heart cytochrome-c oxidase has been obtained by two independent methods, namely by difference spectroscopy between different ligated forms using the magnetisation characteristics (field and temperature dependence) of each feature as a guide to its assignment (Greenwood et al, 1983), and from the MCD of an orientated sample (Greenwood et al, 1988). The Tn5 mutant form of nitrous-oxide reductase is deficient in copper, containing up to two copper atomddimer instead of the eight found in the wild-type enzyme and has been shown to contain only the Cu, centre (Viebrock and Zumft, 1987;Dooley et al, 1991 ;Antholine et al, 1992).…”