Exploring the entrance of proton pathways in cytochrome c oxidase from Paracoccus denitrificans: surface charge, buffer capacity and redox-dependent polarity changes at the internal surface.

Cytochrome c oxidase (CcO), the terminal oxidase of cellular respiration, reduces molecular oxygen to water. The mechanism of proton pumping as well as the coupling of proton and electron transfer is still not understood in this redox-linked proton pump. Eleven residues at the aqueous-exposed surfaces of CcO from Paracoccus denitrificans have been exchanged to cysteines in a two-subunit base variant to yield single reactive cysteine variants. These variants are designed to provide unique labeling sites for probes to be used in spectroscopic experiments investigating the mechanism of proton pumping in CcO. To this end we have shown that all cysteine variants are enzymatically active. Cysteine positions at the negative (N-) side of the membrane are located close to the entrance of the D- and K-proton transfer pathways that connect the N-side with the catalytic oxygen reduction site. Labeling of the pH-indicator dye fluorescein to these sites allowed us to determine the surface potential at the cytoplasmic CcO surface, which corresponds to a surface charge density of -0.5 elementary charge/1000Å(2). In addition, acid-base titrations revealed values of CcO buffer capacity. Polarity measurements of the label environment at the N-side provided (i) site-specific values indicative of a hydrophilic and a more hydrophobic environment dependent on the label position, and (ii) information on a global change to a more apolar environment upon reduction of the enzyme. Thus, the redox state of the copper and heme centers inside the hydrophobic interior of CcO affect the properties at the cytoplasmic surface.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase.

Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-µs electron injection into Cu(A), close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H(+)/COX and 1 H(+)/COX, respectively). The subsequent 10-µs transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ∼0.5 H(+)/COX at this side to electrostatically compensate the charge of the electron. With ∼200 µs, all but 0.4 H(+) at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called "pump site." Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to Cu(A). These results support the idea of a proton-collecting antenna, switched on by electron injection.

Functional interaction structures of the photochromic retinal protein rhodopsin.

We studied functional interaction structures of the vertebrate membrane photoreceptor rhodopsin containing retinal as a chromophore. Using time-resolved fluorescence depolarization we analyzed real-time dynamics and conformational changes of the cytoplasmic helix 8 (H8) preceding the long C-terminal tail of rhodopsin. H8 runs parallel to the membrane surface and extends from transmembrane helix 7 whose highly conserved NPxxY(x)F motif connects that region of rhodopsin with the retinal binding pocket. Our measurements indicate that photo-induced retinal isomerization from 11-cis to all-trans provokes conformational changes of H8, including slower motion and reduced flexibility, that are specific for the active metarhodopsin-II photo-intermediate. These conformational changes are absent in the retinal-devoid state opsin and in the phosphorylated metarhodopsin-II state upon receptor deactivation. Furthermore we show that membrane rim effects can influence interfacial reactions at the cytoplasmic rhodopsin surface such as proton transfer reactions between surface and aqueous bulk phase or binding of the signaling protein transducin visualized with single-molecule widefield microscopy. These findings are important for an understanding of the effects of membrane structure on the photo-transduction mechanism.

Dissection of environmental changes at the cytoplasmic surface of light-activated bacteriorhodopsin and visual rhodopsin: sequence of spectrally silent steps.

The physico-chemical properties as well as the conformation of the cytoplasmic surface of the 7-helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100, 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light-activated bR and rhodopsin detected by time-resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real-time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin-II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time-resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M-intermediate as well as the active metarhodopsin-II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin-II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin-II decay.