Cytochrome c Interaction with Yeast Cytochrome b₂

Abstract

Fluorescent derivatives of cytochrome c were prepared by replacing the heme iron with closed‐shell metals such as zinc or tin. The iron‐free derivatives of cytochrome c bind to yeast lactate dehydrogenase (cytochrome b2) stoichiometrically and with high affinity. Spectral overlap exists between the fluorescence of porphyrin, Zn(II) or Sn(IV) cytochrome c and the absorption of the heme of cytochrome b; therefore dipole‐dipole interaction is possible as predicted by Forster's theory of energy transfer. Changes in the fluorescence yield and the fluorescent decay profile of the cytochrome c derivatives are consistent with the view that the heme distance is sufficiently close for dipolar interactions. The distance calculated from the data depends upon assumptions in the theory for energy transfer and uncertainties in the experiment. It can be argued that due to the symmetry of the metalloporphyrins the relative orientations of the two hemes do not introduce a significant uncertainty in the calculation. However the decay profiles of the iron‐free cytochromes are complex, possibly reflecting structural rearrangement of the polypeptide chain during the fluorescent lifetime. The steady‐state fluorescent yields would indicate that the mean distance is around 1.8 nm

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

“…u / w m a h cytochrome c decays through a single exponential with a lifctime of 2.00 0.05 ns at 10 "C. This lifetime is somewhat shorter (by approx. 40 %) than that reported for horse heart Zn-cytochrome c by Vanderkooi et al [15,221, who used a phase modulation instrument and a deconvolution technique. [26] representing the distribution of the high-molecular-weight component through the distal cell region (slope 1.33, m(npp, = 22400 Da) On the other hand, H .…”

“…anomala. Preliminary titration experiments showed binding stoicliiometries and fluorescence changes widely different from those reported by Vanderkooi et al [15]. It thus appeared worth investigating the molecular size, the homogeneity and the spectral properties of Zn(I1)-substituted H .…”

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

“…These authors made use of the amount of fluorescence quenching to estimate the heme b, to Zn-porphyrin L' distance within the complex, assuming that fluorescence quenching proceeds from resonant (Forster) energy transfer between the related chromophores [18]. The validity of this assumption appears questionable from both theoretical and experimental lines of evidence.…”

Strength in Numbers: Effects of Acceptor Abundance on FRET Efficiency

“…If we denote R n as the Fçrster distance for a FRET system with n acceptors, then by substituting Equation (14) into Equation (10) [see the Experimental and Computational Section], the relationship between R n and R 0 is [Eqs. (4) and (5)]: www.chemphyschem.org…”

“…[2] The rate of transfer shows strong distance dependence (it is inversely proportional to the sixth power of the distance between acceptor and donor) which allows FRET measurements to be used as a spectroscopic ruler for assessing molecular distances. [3][4][5][6][7] Energy transfer can be characterized by the efficiency of the transfer, which gives the probability for the donor excitation energy to be transferred to the acceptor. FRET measurement is a valuable tool for investigating biological processes at the molecular level, because interactions can be imaged for distances far below the diffraction limit of conventional imaging techniques.…”