CYANIDE BINDING AT THE NONHEME FE2-QUINONE COMPLEX OF PHOTOSYSTEM-II - AT HIGH-CONCENTRATIONS, CYANIDE CONVERTS THE FE2+ FROM HIGH (S=2) TOLOW (S=0) SPIN( OF THE IRON)

The primary electron acceptor complex of photosystem II, Q(A)Fe(2+), c an bind a number of small molecules at the iron site, including cyanid e [Koulougliotis, D., Kostopoulos, T., Petrouleas, V., & Diner, B. A. (1993) Biochim. Biophys. Acta 1141, 275-282)]. In the presence of NaCN (30-300 mM) at pH 6.5, the reduced state, Q(A)-Fe2+, produced either by illumination at less than or equal to 200 K or by reduction in the dark with sodium dithionite, is characterized by a g = 1.98 EPR signal . The light- or dithionite-induced g = 1.98 signal decays with increas ing pH above 6.5 and is almost totally absent at pH 8.1 and NaCN conce ntrations above 300 mM. However, at high pH (8.1), the g = 1.98 signal still forms transiently before it decays with a t(1/2) of approximate ly 30 min in spinach BBY preparations treated with 100 mM NaCN. Comple mentary to the disappearance of the g = 1.98 signal with increasing pH or incubation time, a new EPR signal develops at g = 2.0045. This sig nal has the characteristics of the semiquinone, Q(A)(-), uncoupled fro m its magnetic interaction with the iron. Prolonged incubation of a hi gh pH, high cyanide treated sample in a cyanide-free medium at pH 6 re stores the ability of the sample to develop the cyanide-induced g = 1. 98 signal at pH 6.5. This indicates that the iron is not physically di ssociated during the high pH cyanide treatment. The high pH, high cyan ide effects are accompanied by the conversion of the characteristic Fe 2+(S = 2) Mossbauer doublet [isomer shift (Fe) = 1.19 mm/s, quadrupole splitting = 2.95 mm/s] to a new one with parameters (isomer shift = 0 .26 mm/s, quadrupole splitting = 0.36 mm/s) characteristic of an Fe2+( S = 0) state. This explains the loss of the magnetic interaction of Q( A)(-) with the iron. The present results, combined with the earlier st udy, suggest a progressive binding of two or three cyanides at the iro n site. The g = 1.98 to g 2.0045 conversion (reflecting the high- to l ow-spin conversion of the iron) develops as a function of increasing C N- concentration at high pH, with a K-d Of approximately 1.2 mM. If we assume that CN- is the active species for the earlier steps too, the respective Kd)s are 0.1-0.2 mM (development of the g = 1.98 signal) an d 10-20 mu M (competition of cyanide with approximately 300 mu M NO fo r binding to the iron).