SITE-DIRECTED MUTATIONS NEAR THE L-SUBUNIT D-HELIX OF THE PURPLE BACTERIAL REACTION-CENTER - A PARTIAL MODEL FOR THE PRIMARY DONOR OF PHOTOSYSTEM-II

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem IT (PS II) reaction center (RC). To construct this mutant (denoted D1-IL MH), four residues in the bacterial L subunit were mutagenized, such t hat an Ii-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteri ochlorophyll (Bchl) dimer which constitutes the primary donor are subs tantially altered by these modifications, to the degree that the dimer becomes functionally much more ''monomeric''. The changes include (I) an increase in the values of the zero-field splitting (ZFS) parameter s, as measured by electron paramagnetic resonance (EPR), for the spin- polarized triplet state, P-3, from /D/=185 x 10(-1)cm(-1) and /E/=31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/=200 x 10(-4) cm(-1) and /E/=44 x 10(-4) cm(-1) in the mutant and (2) an incr ease in the EPR line width of the oxidized stale, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of P-3 in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potent ial in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV m ore negative) than in WT Rb. capsulatus. In addition, Raman measuremen ts indicate that the hydrogen bond between His(L168) and the C-2 acety l carbonyl oxygen of the Bchl on the active electron transfer pathway (P-A) is absent in the mutant, due to the fact that His(L168) in the W T sequence has been replaced by a leucine in D1-ILMH. However, the Ram an data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C-9 keto carbonyl oxygen of P-A and an unknown hydro gen-bond donor. Thus, although the protein environment around one of t he Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary don or that is oriented like the one in PS II.