Identification of a Novel Protonation Pattern for Carboxylic Acids upon QB Photoreduction in Rhodobacter sphaeroides Reaction Center Mutants at Asp-L213 and Glu-L212 Sites

• Published in: Biochemistry (Easton) 2004-06, Vol.43 (23), p.7236-7243
• Main Authors: Nabedryk, Eliane, Breton, Jacques, Okamura, Melvin Y, Paddock, Mark L
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi049342y

In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule QB. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon formation, QB - induces protonation of Glu-L212, located within 5 Å of QB, resulting in a IR signal at 1728 cm-1. However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770−1700 cm-1). In particular, no signal for Asp-L213 is found despite its juxtaposition to QB and importance for proton uptake on the second electron-transfer step. In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of QB was modified by interchanging Asp and Glu at the L213 and L212 positions. The QB -/QB FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770−1700 cm-1 range shows several distinct new signals, which are sensitive to 1H/2H isotopic exchange, indicating that the reduction of QB results in the change of the protonation state of several carboxylic acids. The new bands at 1752 and 1747 cm-1 were assigned to an increase of protonation in response to QB reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues. Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair. Implications for the native reaction center are discussed.