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Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics.

ABSTRACT: Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm(-1) was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q(A), were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P(L)/P(M))). The region between 1600 and 1800 cm(-1) encompasses absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state P(+)B(L)(-) (where B(L) is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P(+)B(L)(-); the difference spectrum indicates that the 9-keto C=O stretch of B(L) is located around 1670-1680 cm(-1). After subsequent electron transfer to the bacteriopheophytin H(L) in approximately 1 ps, the state P(+)H(L)(-) was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P(+)H(L)(-) radical pair on the approximately 20 ps timescale, accompanied by a change in the relative ratio of the P(L)(+) and P(M)(+) bands and by a minor change in the band amplitude at 1640 cm(-1) that may be tentatively ascribed to the response of an amide C=O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P(+)H(L)(-) is due to an increased localization of the electron hole on the P(L) half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C=O oscillators.

SUBMITTER: Pawlowicz NP

PROVIDER: S-EPMC2479572 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics.

Pawlowicz Natalia P NP van Grondelle Rienk R van Stokkum Ivo H M IH Breton Jacques J Jones Michael R MR Groot Marie Louise ML

Biophysical journal 20080418 3

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm(-1) was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q(A), were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct ex ...[more]

PMID: 18424493

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Project description:The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an approximately 15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the F --> O transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O(2) shows approximately 200-fold inhibition of the F --> O step (k approximately 2 s(-1) at pH 8) which is not compatible with enzyme turnover ( approximately 30 s(-1)). Presumably, an abnormal intermediate F(deprotonated) is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the F --> O transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H(2)O(2), decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Deltapsi generation coupled to the flash-induced F --> O transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a(3) from the Fe(4+) horizontal lineO(2-) state to the Fe(3+)-OH(-) state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the F --> O step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of approximately 10 and approximately 40 mus and a magnitude ratio of approximately 3:2. The 10 mus phase matches vectorial electron transfer from Cu(A) to heme a, whereas the 40 mus component is assigned to intraprotein proton displacement across approximately 20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D-channel mutants as well as with cyanide-inhibited wild-type oxidase. The finding helps to reconcile the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme ( approximately 30%) with the net electrogenicity of the F --> O transition coupled to transmembrane transfer of two charges per electron.

Project description:ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

Dataset Information

Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics.

Publications

Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics.

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