Mutational analysis of the binding sites of the phylloquinones in PS I

Abstract

Due to the symmetry of its heterodimeric core, which is composed of the related PsaA and PsaB membrane polypeptides, photosystem I possesses two cofactor branches that traverse the membrane. There are two phylloquinones in PS I, at least one of which serves as a secondary electron acceptor called A1. Using a combination of site-directed mutagenesis and EPR spectroscopy, we have probed the phylloquinone sites and identified mutants that affect the properties of the A1 cofactor. The PsaA-W693F mutant causes loss of both the photo-accumulated A1- EPR signal and an alteration in the time-resolved P700+ A1- EPR spectrum. Mutation of the corresponding tryptophan in PsaB had no such effect. However, flash absorption spectroscopy of isolated PS I particles revealed that mutation of either tryptophan resulted in an acceleration of the back-reaction from the terminal iron-sulfur clusters. Examination of wild-type Chlamydomonas PS I by in vivo flash spectroscopy demonstrated that reoxidation of A1, as measured by monitoring decay of an absorption increase at 380-400 nm, occurs with biphasic kinetics (half-times of about 13 and 140 ns). The PsaA-W693F mutation specifically affected both the rate of the slower kinetic component (slowed 3-4 fold) and its decay-associated spectrum, while the PsaB-W673F mutation had similar effects on the faster phase. Thus it seems that, although the two phylloquinones of PS I are functionally different, both of them may be used as intermediates in forward and reverse electron transfer through PS I.