Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase

Abstract

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.

Investigation of the electron-transfer properties of cytochrome c oxidase covalently cross-linked to Fe- or Zn-containing cytochrome c

“…In agreement with others [13,14], we find that at low ionic strengths cytochrome c oxidase makes a tight 1: 1 electrostatic complex with its substrate cytochrome c; this complex may be isolated by gel-filtration chromatography and may be dissociated into its separate components at higher ionic strengths ( > 0.1 M) [13]. Addition of EDC to the low-ionic-strength electrostatic complex is known to yield a covalent complex which does not dissociate on addition of salt [15].…”

Anions induce conformational changes and influence the activity and photoaffinity-labelling by 8-azido-ATP of isolated cytochrome c oxidase

“…and c.d. studies of the oxidized and reduced enzyme, and of Alleyne & Wilson (1987) from zinc cytochrome c fluorescence studies. Recently, Scholes & Malmstr6m (1986) suggested that the enzyme underwent a transition from a 'closed' to an 'open' conformation upon reduction.…”

Computational Design of Two-Photon Fluorescent Probes for a Zinc Ion Based on a Salen Ligand

“…In 1987, the first zinc-ion fluorescent probe was discovered, which shows the change of conformation when zinc cytochrome fluorescence acts as a probe in cytochrome oxidase . Also, then many types of Zn 2+ fluorescent probes have been developed in succession; most of them can be applied to detecting Zn 2+ in living cells, − which has made outstanding contributions to the exploration of the functions and properties and the binding mode for the zinc ion in organisms. ,− However, the theoretical research of Zn 2+ fluorescent probes is limited; the primary reason is that the theory is immature and the cognition for the molecular probe is not perfect, so that the theoretical study on probe molecules is just in its infancy.…”