EFFECTS OF HYDROGEN BENDING TO A BACTERIOCHLOROPHYLL-BACTERIOPHEOPHYTIN DIMER IN REACTION CENTERS FROM RHODOBACTER-SPHAEROIDES

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)-bacteriopheophytin (BPhe) dimer with and w ithout hydrogen bonds to the conjugated carbonyl groups. The heterodim er mutation His M202 to Leu was combined with each of the following mu tations: His L168 to Phe, which should remove an existing hydrogen bon d to the BChl molecule; Leu L131 to His, which should add a hydrogen b ond to the BChl molecule; and Leu M160 to His and Phe M197 to His, eac h of which should add a hydrogen bond to the BPhe molecule [Rautter, J ., Lendzian, F., Schulz, C,, Fetsch, A,, Kuhn, M,, Lin, X., Williams, J. C,, Allen, J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that a ll of the mutants contain a heterodimer. The bands in the resonance Ra man spectra arising from the BPhe molecule, which is selectively enhan ced, exhibit the shifts expected for the addition of a hydrogen bond t o the 9-keto and 2-acetyl carbonyl groups. The oxidation-reduction mid point potential of the donor is increased by approximately 85 mV by th e addition of a hydrogen bond to the BChl molecule but is only increas ed by approximately 15 mV by the addition of a hydrogen bond to the BP he molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potenti al. The yield of electron transfer to the primary quinone is 5-fold re duced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the do nor a. band in the mutant that adds a hydrogen bond to the BChl molecu le. The changes in the optical spectra and the yield of electron trans fer are consistent with a model in which the addition of a hydrogen bo nd to the BChl molecule increases the energy of an internal charge tra nsfer state while the addition to the BPhe molecule stabilizes this st ate. The results show that the properties of the heterodimer are diffe rent depending on which side is hydrogen-bonded and suggest that the h ydrogen bonds alter the energy of the internal charge transfer state i n a well-defined manner.