Identification of the proton pathway in bacterial reaction centers: cooperation between Asp-M17 and Asp-L210 facilitates proton transfer to the secondary quinone (QB).
The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.
Paddock
M L
ML
Department of Physics 0319, 9500 Gilman Drive, University of California, San Diego, La Jolla, California 92093, USA.
Adelroth
P
P
Chang
C
C
Abresch
E C
EC
Feher
G
G
Okamura
M Y
MY
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D016227:Benzoquinones / Q000378:metabolism*; D004579:Electron Transport / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010782:Photolysis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides; D055672:Static Electricity
D016227:Benzoquinones; D010455:Peptides; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; C004532:quinone