Departement de Biologie Cellulaire et Moleculaire, CEA CNRS URA 2096,Centre d'Etudes de
Saclay, Gif-sur-Yvette, France. wcoleman@kairos-scientific.com
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 II (PS II) reaction center (RC). To construct
this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized,
such that an 11-residue segment was made identical to the analogous segment from the D1
subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which
constitutes the primary donor are substantially altered by these modifications, to the
degree that the dimer becomes functionally much more "monomeric". The changes
include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as
measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P,
from /D/ = 185 x 10-4 cm-1 and /E/ = 31 x 10-4 cm-1
in wild-type (WT) chromatophore membranes to /D/ = 200 x 10-4 cm-)
and /E/ = 44 x 10-4 cm-1 in the mutant and (2) an increase in the
EPR line width of the oxidized state, 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 3P 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 potential in
PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than
in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen
bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl
on the active electron transfer pathway (PA) is absent in the mutant, due to
the fact that HisL168 in the WT sequence has been replaced by a leucine in
D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the
D1-ILMH RCs, between the C9 keto carbonyl oxygen of PA and an
unknown hydrogen-bond donor. Thus, although the protein environment around one of the
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 donor that is oriented like the one in PS II.