IN BACTERIAL REACTION CENTERS RAPID DELIVERY OF THE 2ND PROTON TO Q(B) CAN BE ACHIEVED IN THE ABSENCE OF L212GLU

Citation
J. Miksovska et al., IN BACTERIAL REACTION CENTERS RAPID DELIVERY OF THE 2ND PROTON TO Q(B) CAN BE ACHIEVED IN THE ABSENCE OF L212GLU, Biochemistry, 36(40), 1997, pp. 12216-12226
Citations number
47
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0006-2960
Volume
36
Issue
40
Year of publication
1997
Pages
12216 - 12226
Database
ISI
SICI code
0006-2960(1997)36:40<12216:IBRCRD>2.0.ZU;2-6
Abstract
In the reaction center (RC) of Rhodobacter capsulatus, residue L212Glu is a component of the pathway for proton transfer to the reduced seco ndary quinone, Q(B). We isolated phenotypic revertants of the photosyn thetically incompetent (PS-) L212Glu-->Gln mutant; all of them retain the L212Glu-->Gln substitution and carry a second-site mutation: L227L eu-->Phe, L228Gly-->Asp, L231Arg-->Cys, or M231Arg-->Cys. We also char acterized the L212Ala strain, which is a phenotypic revertant of the P S- L212Glu-L213Asp-->Ala-Ala mutant. The activities of the RCs of thes e strains-all of which lack L212Glu-were studied by flash-induced abso rption spectroscopy. At pH 7.5, the rate of second electron transfer i n the L212Q mutant is comparable to the wild-type rate. However, this mutant shows a marked decrease in the rate of-cytochrome oxidation und er strong continuous illumination and a very slow phase (0.66 s(-1)) o f the proton transfer kinetics following the second flash, indicating that transfer of the second proton to Q(B) is slowed more than 1000-fo ld. The levels of recovery of the functional capabilities in the rever tant RCs vary widely; their rates of cytochrome oxidation were interme diate between those of the wild-type and the L212Q mutant. The kinetic s of proton transfer following the second flash show a significant rec overy in the L212Q + M231C and L212A RCs (330-540 s(-1)), but the L212 Q + L227F RCs recover this function only partially. Compensation for t he lack of L212Glu in revertant RCs is discussed in terms of (i) confo rmational changes that could allow water molecules to approach closer to Q(B) and/or (ii) the increase in the negative electrostatic environ ment and the resultant I ise in the free energy level of Q(B)(-) that is induced by the mutations. The stoichiometries of H+/Q(B)(-) proton uptake below pH 7.5 in the L212Q, mutant, the L212Q + M231C revertant, and the wild-type strains are essentially equivalent, suggesting that L212Glu is protonated at neutral pH in wild-type RCs. This is also su pported by the P(+)Q(-) charge recombination data. Comparison of H+/Q( B)(-) proton uptake data with those obtained previously for the stoich iometries of H+/Q(A)(-) proton uptake [Miksovska, J., Maroti, P., Tand ori, J., Schiffer, M., Hanson, D. K., Sebban, P. (1996) Biochemistry. 35, 15411-15417] suggests that L212Glu is the key to the electrostatic and perhaps structural interaction between the two quinone sites.