Structure and function of cytochrome c(2) in electron transfer complexes with the photosynthetic reaction center of Rhodobacter sphaeroides: Optical linear dichroism and EPR

The photosynthetic reaction center (RC) and its secondary electron donor the water-soluble cytochrome (cyt) c(2) from the purple bacterium Rhodobacter sphaeroides have been used in cross-linked and non-cross-linked complexes, oriented in compressed gels or partially dried multilayers, to study the respective orientation of the primary donor P (BChl dimer) and of cyt c(2). Three methods were used: (i) Polarized optical absorption spectra at 295 and 10 K were measured and the linear dichroism of the two individual transitions (Q(x), Q(y)), which are nearly degenerate within the alpha-band of reduced cyt c(2), was determined. Attribution of the polarization directions to the molecular axes within the heme plane yielded the average cyt orientation in the complexes. (ii) Time-resolved flash absorption measurements using polarized light allowed determination of the orientation of cyt c(2) in complexes which differ in their kinetics of electron transfer. (iii) EPR spectroscopy of ferricyt c(2) in cross-linked RC-cyt c(2) complexes was used to determine the angle between the heme and the membrane plane. The results suggest the following structural properties for the docking of cyt c(2) to the RC: (i) In cross-linked complexes, the two cytochromes displaying half-lives of 0.7 and 60 mu s for electron transfer to P+ are similarly oriented (difference <10 degrees). (ii) For cross-linked cyt c(2) the heme plane is parallel to the symmetry axis of the RC (0 degrees+/-10 degrees). Moreover, the Q,transition, which is assumed to be polarized within the ring III-ring I direction of the heme plane, makes an angle of 56 degrees+/-1 degrees with the symmetry axis. (iii) The dichroism spectrum for the fast phase (0.7 mu s) for the non-cross-linked cyt c(2)-RC complex suggests an orientation similar to that of cross-linked cyt c(2), but the heme plane is tilted about 20 degrees closer to the membrane, An alternative model is that two or more bound states of cyt c(2) with heme plane tilt angles between 0 degrees and 30 degrees allow the fast electron transfer. Zero-length cross-linking of cyt c(2) may take place in one of these bound states. These orientations of cyt c(2) are compared to different structural models of RC-cyt c(2) complexes proposed previously. The relation of the two kinetic phases observed in cross-linked cyt c(2) complexes to biphasic kinetics of the mobile reaction partners is discussed with respect to the dynamic electrostatic interactions during the formation of a docking complex and its dissociation. A mechanism is proposed in which a pre-orientation of cyt c(2) relative to the membrane plane occurs by interaction of its strong electrostatic dipole with the negative surface charges of the RC. The optimal matching of the oppositely charged surfaces of the two proteins necessitates further rotation of the cyt around its dipole axis.