Mutation induced modulation of hydrogen bonding to P700 studied using FTIR difference spectroscopy.
Site-directed mutagenesis in combination with Fourier transform infrared difference spectroscopy has been used to study how hydrogen bonding modulates the electronic and physical organization of P700, the primary electron donor in photosystem I. Wild-type PS I particles from Chlamydomonas reinhardtii and a mutant in which ThrA739 is changed to alanine [TA(A739) mutant] were studied. ThrA739 is thought to provide a hydrogen bond to the chlorophyll-a' molecule of P700 (the two chlorophylls of P700 (P700(+)) will be called P(A) and P(B) (P(A)(+) and P(B)(+))). The mutation considerably alters the (P700(+)-P700) FTIR difference spectra. However, we were able to describe all of the mutation induced changes in the difference spectra in terms of difference band assignments that were proposed recently (Hastings, G., Ramesh, V. M., Wang, R., Sivakumar, V. and Webber, A. (2001) Biochemistry 40, 12943-12949). Upon comparison of mutant and wild type (P700(+)-P700) FTIR difference spectra, it is shown that (1) the 13(3) ester carbonyl modes of P(A) and P(B) are unaltered upon mutation of ThrA739 to alanine. (2) The 13(3) ester carbonyl modes of P(A)(+)/P(B)(+) upshift/downshift upon mutation. These oppositely directed shifts indicate that the mutation modifies the charge distribution over the pigments in the P700(+) state, with charge on P(B) being relocated onto P(A). We also show that the 13(1) keto carbonyl mode of P(B)/P(B)(+) is unaltered/downshifted upon mutation, as is expected for the above-described mutation induced charge redistribution in P700(+). Although the 13(3) ester carbonyl modes of the chlorophylls of P700 in the ground state are unaltered upon mutation, the 13(1) keto carbonyl mode of P(A) upshifts upon mutation, as does the 13(1) keto carbonyl mode of P(A)(+). For P700 in the ground state, bands that we associate with HisA676/HisB656 upshift/downshift upon mutation. For the P700(+) state, bands that we associate with HisA676/HisB656 also upshift/downshift upon mutation. These observations are also consistent with the notion that the mutation leads to the charge on P(B)(+) being relocated onto P(A)(+). In addition, we suggest that a hydrogen bond to the 13(1) keto carbonyl of P(A) is still present in the TA(A739) mutant, probably mediated through an introduced water molecule.
Wang
Ruili
R
Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303, USA.
Sivakumar
Velautham
V
Li
Yajing
Y
Redding
Kevin
K
Hastings
Gary
G
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D002734:Chlorophyll / Q000737:chemistry* / Q000235:genetics; D002736:Chloroplasts / Q000378:metabolism; D006860:Hydrogen Bonding; D045342:Light-Harvesting Protein Complexes; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D045331:Photosystem I Protein Complex; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011994:Recombinant Proteins; D002734:Chlorophyll; C001785:chlorophyll P 700