Quinone reduction via secondary B-branch electron transfer in mutant bacterial reaction centers.
Symmetry-related branches of electron-transfer cofactors-initiating with a primary electron donor (P) and terminating in quinone acceptors (Q)-are common features of photosynthetic reaction centers (RC). Experimental observations show activity of only one of them-the A branch-in wild-type bacterial RCs. In a mutant RC, we now demonstrate that electron transfer can occur along the entire, normally inactive B-branch pathway to reduce the terminal acceptor Q(B) on the time scale of nanoseconds. The transmembrane charge-separated state P(+)Q(B)(-) is created in this manner in a Rhodobacter capsulatus RC containing the F(L181)Y-Y(M208)F-L(M212)H-W(M250)V mutations (YFHV). The W(M250)V mutation quantitatively blocks binding of Q(A), thereby eliminating Q(B) reduction via the normal A-branch pathway. Full occupancy of the Q(B) site by the native UQ(10) is ensured (without the necessity of reconstitution by exogenous quinone) by purification of RCs with the mild detergent, Deriphat 160-C. The lifetime of P(+)Q(B)(-) in the YFHV mutant RC is >6 s (at pH 8.0, 298 K). This charge-separated state is not formed upon addition of competitive inhibitors of Q(B) binding (terbutryn or stigmatellin). Furthermore, this lifetime is much longer than the value of approximately 1-1.5 s found when P(+)Q(B)(-) is produced in the wild-type RC by A-side activity alone. Collectively, these results demonstrate that P(+)Q(B)(-) is formed solely by activity of the B-branch carriers in the YFHV RC. In comparison, P(+)Q(B)(-) can form by either the A or B branches in the YFH RC, as indicated by the biexponential lifetimes of approximately 1 and approximately 6-10 s. These findings suggest that P(+)Q(B)(-) states formed via the two branches are distinct and that P(+)Q(B)(-) formed by the B side does not decay via the normal (indirect) pathway that utilizes the A-side cofactors when present. These differences may report on structural and energetic factors that further distinguish the functional asymmetry of the two cofactor branches.
Laible
Philip D
PD
Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, USA. laible@anl.gov
Kirmaier
Christine
C
Udawatte
Chandani S M
CS
Hofman
Samuel J
SJ
Holten
Dewey
D
Hanson
Deborah K
DK
D019943:Amino Acid Substitution / Q000235:genetics; D001429:Bacteriochlorophylls / Q000378:metabolism; D016227:Benzoquinones / Q000378:metabolism*; D004579:Electron Transport / Q000235:genetics; D004734:Energy Metabolism / Q000235:genetics; D007096:Imidoesters / Q000378:metabolism; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010674:Pheophytins / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000138:chemical synthesis; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D017186:Titrimetry; D014227:Triazines / Q000378:metabolism
D001429:Bacteriochlorophylls; D016227:Benzoquinones; D007096:Imidoesters; D045342:Light-Harvesting Protein Complexes; D010674:Pheophytins; D045322:Photosynthetic Reaction Center Complex Proteins; D014227:Triazines; C034266:N-lauryl iminodipropionate; C006678:bacteriopheophytin; C004532:quinone; C010347:terbutryne