SPIN LABEL MOTION IN THE INTERNAL AQUEOUS COMPARTMENT OF SPINACH THYLAKOIDS

We have measured the motion of the spin label TEMPAMINE (2,2,6,6-tetramethyl piperidine-N-oxyl-4-amine) in the internal aqueous compartment of spinach thylakoids by using potassium ferricyanide (80 mm) to remove TEMPAMINE signals eminating from the external aqueous regions. We found (1): that ferricyanide does not inhibit phosphorylation or electron transport at the concentrations required for TEMPAMINE broadening, but TEMPAMINE acts as a potent uncoupler of electron transport; (2) that TEMPAMINE does not bind detectably to the thylakoid membrane or thylakoid components during the time course of a typical electron spin resonance experiment, but that some binding does occur over a 48-h period to intact thylakoids; (3) that tightly packed intact thylakoids or thylakoids which have been disrupted in a 20% Triton X-100 do not hinder the motion of TEMPAMINE by more than a factor of 1.9; (4) that TEMPAMINE in the presence of 80 mm potassium ferricyanide gives rise to a signal characteristic of TEMPAMINE tumbling isotropically in an aqueous environment with a bulk viscosity of about 10 cP; and (5) that, although ferricyanide leaks slowly into the thylakoid interior, it does not alter the measurement of TEMPAMINE rotation. We conclude that the thylakoid interior is more viscous than bulk water. This may have functional significance regarding transport of electrons from Photosystem II and I to the ATP synthetase. © 1979.