A mechanistic principle for proton pumping by cytochrome c oxidase

Photosynthetic oxygen production by photosystem II (PSII) is responsible for the maintenance of aerobic life on earth. The production of oxygen occurs at the PSII oxygen-evolving complex (OEC), which contains a tetranuclear manganese (Mn) cluster. Photo-induced electron transfer events in the reaction center lead to the accumulation of oxidizing equivalents on the OEC. Four sequential photooxidation reactions are required for oxygen production. The oxidizing complex cycles among five oxidation states, called the S n states, where n refers to the number of oxidizing equivalents stored. Oxygen release occurs during the S 3-to-S0 transition from an unstable intermediate, known as the S4 state. In this report, we present data providing evidence for the production of an intermediate during each S state transition. These proteinderived intermediates are produced on the microsecond to millisecond time scale and are detected by time-resolved vibrational spectroscopy on the microsecond time scale. Our results suggest that a protein-derived conformational change or proton transfer reaction precedes Mn redox reactions during the S 2-to-S3 and S 3-to-S0 transitions.manganese cluster ͉ photosynthesis ͉ photosystem II ͉ time-resolved IR ͉ water oxidation T ime-resolved vibrational spectroscopy can detect chemical intermediates formed during enzymatic catalysis. Advantages include the technique's exquisite structural sensitivity and its high temporal resolution. Recent advances in methodology and interpretation have produced insights into the catalytic mechanism in several biological systems (for examples, see refs. 1-4).In this paper, we report the use of time-resolved IR spectroscopy to investigate the mechanism of photosynthetic water oxidation. Photosystem II (PSII) catalyzes the oxidation of water and the reduction of bound plastoquinone. Photoexcitation of PSII leads to the oxidation of the chlorophyll donor, P 680 , and the sequential reduction of a pheophytin (Fig. 1A, reaction 1) and a plastoquinone, Q A ( Fig. 1 A, reaction 2), in picoseconds. Q A reduces Q B to generate a semiquinone radical, Q B Ϫ , on the microsecond time scale (Fig. 1 A, reaction 3 ) (reviewed in ref. 5).A second photoexcitation leads to the reduction and protonation of Q B Ϫ to form the quinol Q B H 2 . The rate of reduction of Q B is faster than the rate of reduction of Q B Ϫ (see ref. 6 and references therein), which gives rise to a characteristic period-2 oscillation in kinetics originating on the PSII acceptor side (7).The primary chlorophyll donor, P 680 , oxidizes a tyrosine, Y Z (Y161 in the D1 subunit), on the nanosecond to microsecond time scale (Fig. 1 A, reaction 4). In turn, tyrosine Y Z ⅐ oxidizes the oxygen-evolving complex (OEC) on every flash (Fig. 1 A, reaction 5) (8). Four sequential photooxidation reactions are required for oxygen production, and the oxidizing complex cycles among five oxidation states, called the S n states, where n refers to the number of oxidizing equivalents stored (9). The rate of OEC oxidation slows as o...

Section: Discussionmentioning

confidence: 99%

Time-resolved vibrational spectroscopy detects protein-based intermediates in the photosynthetic oxygen-evolving cycle

Barry

Cooper²,

Riso³

et al. 2006

“…Furthermore, the data indicate that the mode of protonation of a membrane-associated proton acceptor can switch from a membrane-promoted to a direct exchange with the bulk solution. This protonation mechanism is likely to be a general feature also for a wide range of membrane-associated proton transporters, such as cytochrome c oxidase, photosynthetic reaction centers, and bacteriorhodopsin (9,15,30) in living cells.…”

Section: Discussionmentioning

confidence: 99%

Surface-coupled proton exchange of a membrane-bound proton acceptor

Sandén

Salomonsson

Brzezinski

et al. 2010

Self Cite

Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pHsensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pK a s, revealed two distinct proton exchange regimes. At high pH (>8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (<7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.biomembrane | diffusion | electrostatic potential | fluorescence correlation spectroscopy (FCS) | proton transfer E nergy conversion in living cells typically involves proton translocation across a membrane, via proton transporters. These transporters maintain a proton electrochemical gradient utilizing free energy provided, for example by electron transfer or light. The free energy stored in this gradient is used, e.g., for transmembrane transport, motility, or synthesis of ATP by the ATP synthase. Results from a range of studies indicate that the membrane plays an important role in these processes, in addition to serving as a barrier. The membrane surface may also provide a proton link between the various membrane-embedded proteins, where the mere two-dimensional confinement of the reactants can also play a role. Enhancement of reaction rates between solute molecules and their target molecules on the surface and in the vicinity of biological membrane interfaces (e.g. ligand binding to membrane proteins) has been demonstrated in several studies and explained in terms of initial nonspecific binding of the solute molecules to the membrane followed by diffusion along the surface to their target molecules (1-8). Studies on some specific membrane-bound proton pumps, for example cytochrome c oxidase or bacteriorhodopsin (9-12), have revealed higher than diffusion-limited rates of proton upta...

“…This reaction can be studied by time-resolved spectroscopy using various detection techniques, which has provided detailed information about the catalytic cycle of the A-type HCuOs (see, e.g., refs. [22][23][24][25][26].…”

mentioning

confidence: 99%

Entrance of the proton pathway in cbb ₃ -type heme-copper oxidases

Lee

Gennis²,

Ädelroth

2011

Self Cite

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O 2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb 3 ) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb 3 numbering) in the accessory subunit CcoP (E25 P ). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb 3 by O 2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25 P as the entry point to the proton pathway in cbb 3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O 2 reduction. The cbb 3 is in addition to O 2 reduction capable of NO reduction, an activity that was largely retained in the E25 P variants, consistent with a scenario where NO reduction in cbb 3 uses protons from the periplasmic side of the membrane.glutamate | nitric oxide | oxygen reduction