Study of the Hansenula anomala yeast flavocytochrome‐b₂–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

Abstract

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn‐cytochrome c appears to be in the form of a stable though non‐covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal‐free porphyrin‐cytochrome c, the precursor of Zn‐cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate‐reduced state showed a simple binding equilibrium (Kd=0.1 μM at I=0.03 M, 10°C) with a stoichiometry of one Zn‐cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn‐porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.

Study of the Hansenula anomala yeast flavocytochrome‐b2‐cytochrome‐c complex

“…Preparation of flavocytochrome b, and cytochrome c. from Hunsenula anomala yeast, Zn,+-substitution of cytochrome c, and static fluorescence measurements, were carried out a s described in the preceding paper [7]. Buffers Fla~~orlehydrogeiia.sc.…”

“…Therefore, the system was not saturated or hindered by multiphotonic (non-linear) processes. Shifting the excitation wavelength to 543.5 nm, corresponding to a maximum in the visible absorption spectrum of Zn-cytochrome c [7], produced no change in either the fluorescence lifetime or intensity normalized for sample absorbance and laser pulse energy. The first harmonic of the neodynium laser at 532 nm was, therefore, used throughout for excitation of Zn-cytochrome c.…”

“…Aliquots of flavocytochrome b, or of the isolated xT, q or FDH,, fragments, stored in cold lactate-containing buffer, were introduced under gentle stirring to a 1 pM solution of the Hunsenula anornala Zn-cytochrome c dimer [7] in 10mM phosphate, 10 mM DL-lactate buffer at 10 ' C , directly in a semimicro cuvette placed in the sample compartment of the fluorimeter. The buffer contained enough lactate to ensure complete reduction of the prosthetic groups in the FDH,., tetramer as well as in flavocytochrome b,.…”

“…The reduction of cytochrome c occurs within a flavocytochrome-h, -cytochrome-c complex [4, 51. This complex is stable at low ionic strength with the stoichiometry of one molecule of cytochrome c bound per molecule of Hunsenula anomalu flavocytochrome h, protomer [6,7]. This suggests that each flavocytochrome b2 protomer may offer specific association areas for tight cytochrome c binding.…”

“…anornufa cytochrome c dimer characterized in the preceding paper [7] to determine the cytochrome c binding affinity of some purified proteolytic fragments of flavocytochrome b,. The following fragments were investigated.…”