Stoichiometry Of Proton Release Research Articles

Proton transfer in the photoreceptors phytochrome and photoactive yellow protein

Light-induced activation of the photoreceptors phytochrome and photoactive yellow protein (PYP) is accompanied by protonation changes of the respective chromophores and key residues in the protein moiety. For both systems, proton exchange with the external medium could be observed with pH electrode measurements and with UV-visible absorption spectroscopy using appropriate pH indicator dyes. From these signals, the stoichiometry of proton release and uptake, respectively, was determined by different calibration procedures which will be presented and discussed. Kinetic information on these processes is only available from time-resolved measurements with pH indicator dyes. Vibrational spectroscopy methods such as Fourier transform infrared spectroscopy and resonance Raman scattering provided information on the protonation state of individual functional groups suggesting that internal proton transfer processes are involved as well. Deuterium kinetic isotope effects that occurred in the Pr --> Pfr phototransformation of the bacteriophytochromes Cph1 and Agp1 were consistent with proton transfer reactions as rate-limiting steps. In contrast, the apparent rate constants in the photocycle of PYP exhibited only small kinetic isotope effects that could not be interpreted conclusively. Possible mechanisms of proton transfer in the activation of phytochrome and PYP will be discussed.

Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues.

Transition metal ions bind to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides and slow the light-induced electron and proton transfer to the secondary quinone, Q(B). We studied the properties of the metal ion-RC complex by measuring the pH dependence of the dissociation constant and the stoichiometry of proton release upon ligand formation. We investigated the mechanism of inhibition by measuring the stoichiometry and kinetics of flash-induced proton binding, the transfer of (first and second) electrons to Q(B), and the rate of steady-state turnover of the RC in the absence and presence of Cd(2+) and Ni(2+) on a wide pH range. The following results were obtained. (1) The complexation of transition metal ions Cd(2+) and Ni(2+) with the bacterial RC showed strong pH dependence. This observation was explained by different (pH-dependent) states of the metal-ligand cluster: the complex formation was strong when the ligand (Asp and His residues) was deprotonated and was much weaker if the ligand was partly (or fully) protonated. A direct consequence of the model was the pH-dependent proton release upon complexation. (2) The retardation of transfer of electrons and protons to Q(B) was also strongly pH-dependent. The effect was large in the neutral pH range and decreased toward the acidic and alkaline pH values. (3) Steady-state turnover measurements indicated that the rate of the second proton transfer was much less inhibited than that of the first one, which became the rate-limiting step in continuous turnover of the RC. (4) Sodium azide partly recovered the proton transfer rate. The effect is not due to removal of the bound metal ion by azide but probably by formation of a proton-transporting azide network similarly as water molecules may build up proton pathways. (5) We argue that the inhibition comes mainly from pK(a) shifts of key protonatable residues that control the proton transfer along the H-bond network to Q(B). The electrostatic interaction between the metal ion and these residues may result in acidic pK(a) shifts between 1.5 and 2.0 that account for the observed retardation of the electron and proton transfer.

Stoichiometry of Proton Release from the Catalytic Center in Photosynthetic Water Oxidation: REEXAMINATION BY A GLASS ELECTRODE STUDY AT pH 5.5–7.2

The catalytic center (CC) of water oxidation in photosystem II passes through four stepwise increased oxidized states (S(0)-S(4)) before O(2) evolution takes place from 2H(2)O in the S(4) --> S(0) transition. The pattern of the release of the four protons from the CC cannot be followed directly in the medium, because proton release from unknown amino acid residues also takes place. However, pH-independent net charge oscillations of 0:0:1:1 in S(0):S(1):S(2):S(3) have been considered as an intrinsic indicator for the H(+) release from the CC. The net charges have been proposed to be created as the charge difference between electron abstraction and H(+) release from the CC. Then the H(+) release from the CC is 1:0:1:2 for the S(0) --> S(1) --> S(2) --> S(3) --> S(0) transition. Strong support for this conclusion is given in this work with the analysis of the pH-dependent pattern of H(+) release in the medium measured directly by a glass electrode between pH 5.5 and 7.2. Improved and crystallizable photosystem II core complexes from the cyanobacterium Synechococcus elongatus were used as material. The pattern can be explained by protons released from the CC with a stoichiometry of 1:0:1:2 and protons from an amino acid group (pK approximately 5.7) that is deprotonated and reprotonated through electrostatic interaction with the oscillating net charges 0:0:1:1 in S(0):S(1):S(2):S(3). Possible water derivatives that circulate through the S states have been named.

ANOTHER ROLE OF CHLOROPHYLL a/b BINDING PROTEINS OF HIGHER PLANTS: THEY MODULATE PROTOLYTIC REACTIONS ASSOCIATED WITH PHOTOSYSTEM II

AbstractChlorophyll alb binding proteins serve as light‐harvesting antennae. Additionally they seem to modulate proton pumping by photosystem II, because their covalent modification by N, N‐dicyclohexylcarbodiimide is paralleled by a short‐circuit of this activity. This side action was further characterized in comparative study with control thylakoids and thylakoids lacking chlorophyll alb binding proteins. The latter were derived from peas grown under intermittent light. They differed from controls in the following: (i) after incubation with A^A^‐dicyclohexylcarbodiimide there was no protonic short circuit, (ii) under flashing light the rate of proton consumption at the acceptor side of photosystem II was accelerated and (iii) the periodical pattern of proton release from water oxidation was flattened out. It was obvious that chlorophyll alb binding proteins modulated the kinetics and the stoichiometry of proton release from water oxidation and proton uptake at the quinone binding pocket.