Kinetic characteristics of formatc/formic acid binding at the plastoquinone reductase site in spinach thylakoids

Oxida t ion of the reduced p r i m a r ) e lec t ron acceptor , Q~-, of Pho tosys tem !I (PS I!) in f e rma te t r e a t ed sp inach thylakoids , was inhib i ted more af te r the second than af te r the f irst ac t in ic f lash. This ind ica tes a slowing of e lec t ron flow on the acceptor side of PS l i from Q/~ to Qf f , the semiqu inone to rm of the secondary p l a s toqu inone aeceptor , formed by electron t r ans fe r af ter the f irst f lash. A hypothes is of e lec t ron t r ans fe r on the accep to r s ide of PS 11 is p roposed to accommoda te the b ica rbona te reve r s ib le f o r m a t e / f o r m i c ac id inh ib i t ion of e lec t ron t r a n s f e r a f t e r single tu rnover f lashes. We suggest tha t the large inh ib i t ion in QA oxida t ion a f te r the second f lash reflects a blockage of the pro ton up take tha t s tabi l izes Q ~ . Kinet ics of onset of inh ib i t ion following fo rmate add i t i on were followed by measu r ing the chlorophyl l a f luorescence yield, ref lect ing the concen t ra t ion of Q A , I ms a f te r the second act in ic f lash as a function of t ime a f te r the add i t i on of formate . The a p p a r e n t ra te cons tan t s for b ind ing a n d unbinding , and the d issoc ia t ion cons t an t of fo rmate were de t e rmine d in the pH range f rom 5.5. to 7.5. The ra te of onset of inhibi t ion following formate add i t ion , ref lect ing fo rmate o r formic ac id b ind ing , was highly d e p e n d e n t on the med ium pH. Measu remen t s on the in i t ia l b ind ing ra te , when one of the two (HCO z / H C O O H ) equ i l ib r ium species was kept cons tant and the o the r var ied, suggested tha t formic acid is the b ind ing species. Th is conc lus ion was consis tent with the observed pH dependence of fo rmate b ind ing .