Hanna Smrz and R.J.Pace
Department of Chemistry, Faculty of Science, Australian National University
There has been much controversy regarding the orientation of the Rieske Iron-Sulfur protein within the thylakoid membrane of plant chloroplasts. It is one of four proteins in the bf complex involved in the electron transfer and proton translocation through the thylakoid membrane, during photosynthesis. A knowledge of the arrangement of the proteins, is important for an understanding of these events. The protein consists of one membrane spanning helix, and the conventional view is that its C-Terminus and Iron-Sulfur cluster are located on the inner, lumenal side of the membrane. The currently accepted mechanism of electron and proton transport through the bf complex, is described by a model known as the Modified Q-Cycle. However, it assumes the conventional orientation of the Rieske FeS protein. Other models need to be considered if the orientation is opposite to that which is conventional.However, some studies have indicated an opposite orientation (1). This report demonstrates the use of Electron Paramagnetic Resonance (E.P.R.) to determine the location of the Rieske Iron-Sulfur centre, and the results indicate an unconventional orientation of the protein.
The Rieske FeS centre has a characteristic E.P.R. signal (when reduced) at g=1.90. Relaxation enhancement studies have been used to calculate approximate distances of paramagnetic centres from a relaxing agent, a dysprosium EDTA complex (Dy[EDTA]- ). Previous studies (eg. 2) on the Rieske FeS cluster have found it to be located approximately 14 Angstroms from the p-side (analogous to the inner lumenal side). However, they have assumed an r-6 dependence (r is the distance between the probe and the centre), on half power saturation. This is applicable only if there is a specific binding site for the Dy[EDTA]- complex, on the protein surface. A recent study (3) has shown that an r-4 (even surface binding) or r-3 (random distribution) dependence is more appropriate. Also, the previous studies were performed on bacterial mitochondria, which have several large extra-membrane proteins on the m-side (analogous to the outer, stromal side), thus preventing close approach of the relaxing agent.
This study was performed on plant thylakoid membranes, and an r-4 dependence has been used to calculate distances. An approximate distance of 14 Angstroms has been calculated, from the outer, stromal side. Vesicles of thylakoid membrane were prepared, and the inside out vesicles were compared to the right side out vesicles. It was found that the right side out vesicles gave a much higher relaxation enhancement effect of the Rieske FeS cluster, compared with the inside out vesicles.
(1) R.T. Theiler and R.A. Neiderman, Localisation of chromatophore Proteins of Rhodobacter sphaeroides, The Journal of Biological Chemistry, 226, 23163-23168, (1991).
(2) R.C.Prince, The Location, Orientation and stoichiometry of the Rieske Iron-Sulfur Cluster in Membranes from Rhodopseudomonas Sphaeroides, B.B.A, 723, 133-138, (1983)
(3) J.B. Innes and G.W. Brudvig, Location and Magnetic Relaxation Properties of the Stable Tyrosine Radical in Photosystem II, Biochemistry, 28, No. 3, 1117-1125, (1989).