Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases proton uptake by Glu-L212 upon first electron transfer to QB in reaction centers from Rhodobacter sphaeroides.
In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located approximately 5 A from the center of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 play an important role in the proton transfer to the Q(B) site [Paddock, M. L., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. (2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the amplitude of the proton uptake by Glu-L212 at pH 8 is increased in the single and double mutant RCs, as is evident from the larger intensity (by 35-55%) of the carboxylic acid band at 1727 cm(-1) in the Q(B)(-)/Q(B) difference spectra of mutant RCs, compared to that at 1728 cm(-1) in native RCs. This implies that the extent of ionization of Glu-L212 in the Q(B) ground state is greater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no changes in the protonation state or the environment of these two residues are detected upon Q(B) reduction. The absence of the 1727 cm(-1) signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 cm(-1) probes the protonation of Glu-L212 in native and mutant RCs.
Nabedryk
E
E
Section de Bioénergétique, CEA-Saclay, 91191 Gif-sur-Yvette, France.
Breton
J
J
Okamura
M Y
MY
Paddock
M L
ML
D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D017666:Deuterium Oxide / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011809:Quinones / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D011809:Quinones; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D017666:Deuterium Oxide