Proton and electron transfer to the secondary quinone (QB) in bacterial reaction centers: the effect of changing the electrostatics in the vicinity of QB by interchanging asp and glu at the L212 and L213 sites.
The bacterial reaction center (RC) plays a central role in photosynthetic energy conversion by facilitating the light induced double reduction and protonation of a bound quinone molecule, QB. Two carboxylic acid residues, Asp-L213 and Glu-L212, located near QB, were previously shown to be important for proton transfer to QB. In this work, the ability of Glu to substitute for Asp at L213 and Asp to substitute for Glu at L212 was tested by site-directed mutagenesis. Both single mutants and a double mutant in which Asp and Glu were exchanged between the two sites were constructed. The electron transfer rate constants kBD (D+QAQB- --> DQAQB), and kAB(2) (DQA-QB- + H+ --> DQA(QBH)-), that are known to be sensitive to the energy of the QB- state, were found to be altered by Asp/Glu substitutions. Both rates were fastest ( approximately 10-fold) in RCs with Asp at both sites, slowest with Glu at both sites ( approximately 50-fold) and relatively unchanged by the caboxylic acid exchange. These changes could be explained if Asp was predominantly ionized and Glu was predominantly protonated at both sites (pH 7.5). The charge recombination kBD suggests an observed approximately 5 pKa unit difference of Glu over Asp. Modeling of kBD by strong electrostatic interactions ( approximately 3-4 pKa units) among negatively charged acids and QB- indicated a lower intrinsic pKa for Asp compared to Glu at either site of approximately 2-3 units. The mechanism of the kAB(2) reaction was determined to be the same in all mutant RCs as for native RCs. A quantitative explanation of the effect of the electrostatic environment on kAB(2) was obtained using the two-step model proposed for native RCs [Graige, M. S., Paddock, M. L., Bruce, J. M., Feher, G., & Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016] which involves fast protonation of the semiquinone followed by rate-limiting electron transfer. Using simple models for the quinone/quinol conversion rate, it is shown that the optimal electrostatic potential for the QB site is close to that found in native RCs.
Paddock
M L
ML
Department of Physics, 0319, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0319, USA.
Feher
G
G
Okamura
M Y
MY
D001224:Aspartic Acid / Q000235:genetics; D004579:Electron Transport; D018698:Glutamic Acid / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D011522:Protons; D012242:Rhodobacter sphaeroides; D055672:Static Electricity; D013347:Subcellular Fractions / Q000378:metabolism; D013816:Thermodynamics; D014451:Ubiquinone / Q000378:metabolism*
D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D014451:Ubiquinone; D001224:Aspartic Acid; D018698:Glutamic Acid