Steric barrier to bathorhodopsin decay in 5-demethyl and mesityl analogues of rhodopsin.

Absorbance difference spectra were recorded from 20 ns to 1 micros after 20 degrees C photoexcitation of artificial visual pigments derived either from 5-demethylretinal or from a mesityl analogue of retinal. Both pigments produced an early photointermediate similar to bovine bathorhodopsin (Batho). In both cases the Batho analogue decayed to a lumirhodopsin (Lumi) analogue via a blue-shifted intermediate, BSI, which formed an equilibrium with the Batho analogue. The stability of 5-demethyl Batho, even though the C8-hydrogen of the polyene chain cannot interact with a ring C5-methyl group to provide a barrier to Batho decay, raises the possibility that the 5-demethylretinal ring binds oppositely from normal to form a pigment with a 6-s-trans ring-chain conformation. If 6-s-trans binding occurred, the ring C1-methyls could replace the C5-methyl in its interaction with the chain C8-hydrogen to preserve the steric barrier to Batho decay, consistent with the kinetic results. The possibility of 6-s-trans binding for 5-demethylretinal also could account for the unexpected blue shift of 5-demethyl visual pigments and could explain why 5-demethyl artificial pigments regenerate so slowly. Although the mesityl analogue BSI's absorption spectrum was blue-shifted relative to its pigment spectrum, the blue shift was much smaller than for rhodopsin's or 5-demethylisorhodopsin's BSI. This suggests that increased C6-C7 torsion may be responsible for some of BSI's blue shift, which is not the case for mesityl analogue BSI either because of reduced spectral sensitivity to C6-C7 torsion or because the symmetry of the mesityl retinal analogue precludes having 6-s-cis and 6-s-trans conformers. The similarity of the mesityl analogue BSI and native BSI lambda(max) values supports the idea that BSI has a 6-s angle near 90 degrees, a condition which could disconnect the chain (and BSI's spectrum) from the double bond specifics of the ring.

The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.

Near-infrared time-resolved optical absorption studies of the reaction of fully reduced cytochrome c oxidase with dioxygen.

Electron transfer during the reaction of fully reduced bovine heart cytochrome oxidase with dioxygen has been studied at 24 degrees C in the near-infrared region following photolysis of the fully reduced CO-bound complex. The transient spectral changes and kinetics were followed on microsecond to millisecond time scales at nine different wavelengths between 597 and 935 nm and were analyzed using singular value decomposition and global exponential fitting. Four apparent lifetimes, 14 micros, 40 micros, 86 micros, and 1.1 ms, were resolved. The near-infrared spectra of the intermediates are extracted on the basis of a previously proposed mechanism [Sucheta et al. (1998) Biochemistry 37, 17905-17914] and compared to model spectra of the postulated intermediates. The data provide a comprehensive picture of the spectral contributions of the different redox centers in their respective oxidation or ligation states in the near-infrared region and strongly support that Cu(A) is partially (2/3), but not fully, oxidized in the 3-electron-reduced ferryl intermediate.

Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection.

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.

Multicolored protein conformation states in the photocycle of transducer-free sensory rhodopsin-I.

Sensory rhodopsin-I (SRI), a phototaxis receptor of archaebacteria, is a retinal-binding protein that exists in the cell membrane intimately associated with a signal-transducing protein (HtrI) homologous to eubacterial chemotaxis receptors. Transducer-free sensory rhodopsin-I (fSRI), from cells devoid of HtrI, undergoes a photochemical cycle kinetically different from that of native SRI. We report here on the measurement and analysis of the photochemical kinetics of fSRI reactions in the 350-750-nm spectral range and in a 10(-7) s to 1 s time window. The lack of specific intermolecular interactions between SRI and HtrI results in early return of the ground form via distinct branching reactions in fSRI, not evident in the photocycle of native SRI. The chromophore transitions are loosely coupled to protein structural transitions. The coexistence of multiple spectral forms within kinetic intermediates is interpreted within the concept of multicolored protein conformational states.

New photointermediates in the two photon signaling pathway of sensory rhodopsin-I.

Sensory rhodopsin-I (SRI) functions as a color discriminating receptor in halobacterial phototaxis. SRI exists in the membrane as a molecular complex with a signal transducer protein. Excitation of its thermally stable form, SRI(587), generates a long-lived photointermediate of its photocycle, S(373), and an attractant phototactic response. S(373) decays thermally in a few seconds into SRI(587.) However, when S(373) is excited by UV-blue light, it photoconverts into SRI(587) in less than a second, generating a repellent phototactic response. Only one intermediate of this back-photoreaction, S(b)(510), is known. We studied the back-photoreaction in both native SRI and its transducer free form fSRI by measuring laser flash induced absorption changes of S(373) photoproducts from 100 ns to 1 s in the 350-750 nm range. Using global exponential fitting, we determined the spectra and kinetics of the photointermediates. S(373) and fS(373) when pumped with 355 nm laser light generate in less than 100 ns two intermediate species: a previously undetected species that absorbs maximally at about 410 nm, S(b)(410), and the previously described S(b)(510). These two intermediates appear to be in a rapid equilibrium, which probably entails protonation change of the Schiff base chromophore. At pH 6 this system relaxes to SRI(587) via another intermediate absorbing maximally around 550 nm, which thermally decays back to the ground state. The same intermediates are seen in the presence and absence of transducer; however, the kinetics are affected by binding of the transducer.

A new approach for studying fast biological reactions involving dioxygen: the reaction of fully reduced cytochrome c oxidase with O2.

We describe a new method for studying rapid biological reactions involving dioxygen. This approach is based on the photolysis of a synthetic caged dioxygen carrier, which produces dioxygen on a fast time scale. The method was used to investigate the reduction of dioxygen to water by cytochrome c oxidase at room temperature following photolysis of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)c obalt(III)] complex. The fact that dioxygen is generated in situ on a nanosecond or faster time scale avoids potential complications related to the fate of photodissociated CO in a conventional CO flow-flash experiment. The cobalt complex is stable at room temperature under anaerobic conditions and releases dioxygen upon irradiation at 355 nm with a quantum yield of 0.04. The complex does not react with reduced cytochrome oxidase or its reducing agents within the mixing time of the experiment, and its photoproducts do not interfere with the kinetics of the dioxygen reduction. The oxidation of the reduced cytochrome oxidase was monitored between 500 and 750 nm using a gated optical spectrometric multichannel analyzer following photodissociation of the cobalt complex. The data were analyzed using singular value decomposition and global exponential fitting, and two apparent lifetimes (380 +/- 50 micros and 1.7 +/- 0.2 ms) were resolved and compared to results from a conventional CO flow-flash experiment. The results show that approximately 90 microM dioxygen can be generated upon a single laser pulse and that this approach can be used to study other fast biological reactions involving O(2).

Effect of NADPH on formation and decay of human metarhodopsin III at physiological temperatures.

Difference absorption spectra were recorded during the formation and decay of metarhodopsin III after sonicated membrane suspensions of rhodopsin were bleached at 37 degrees C. The data were analyzed using SVD, spectral decomposition and global exponential fitting. By comparison of the results in the presence or absence of 70 microM NADPH and those for bovine or human rhodopsin, a single comprehensive scheme was fit to all the data, including reduction of retinal to retinol by the intrinsic retinol dehydrogenase. On the time scale studied the mechanism involves two 382 nm absorbing species and two 468 nm, absorbing species, supporting the notion that human metarhodopsin III is not a homogeneous species. The results confirm that metarhodopsin III forms and persists sufficiently long in the human retina under physiological conditions that it could undergo secondary photoisomerization.

Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

Structural constraints imposed by a non-native disulfide cause reversible changes in rhodopsin photointermediate kinetics.

Suspensions of bovine rhodopsin in 2% lauryl maltoside detergent were treated with Cu(phen)(3)(2+) to form a disulfide bridge between cysteines 140 and 222 which occur naturally in the bovine rhodopsin sequence. Absorption difference spectra were collected after excitation with a pulse of 477 nm light on the time scale from 1 micros to 690 ms, and the results were analyzed using global exponential fitting. Only two exponentials could be fit to data from the Cu(phen)(3)(2+)-treated rhodopsin, while three exponentials were needed to fit data either from untreated rhodopsin or from Cu(phen)(3)(2+)-oxidized rhodopsin after further dithiothreitol reduction. Dithiothreitol treatment of rhodopsin which had not been previously oxidized with Cu(phen)(3)(2+) had no effect on the observed kinetics. Since the 140-222 disulfide has previously been shown to block transducin activation, its effects on rhodopsin activation are of considerable interest. Cu(phen)(3)(2+) treatment favors formation of the meta I(380) intermediate relative to meta I(480) and slows formation of meta II from meta I(380). This suggests that the protein change involved in meta I(380) formation is similar to the structural constraint introduced by the 140-222 disulfide. These results show that formation of disulfides in rhodopsin has potential as a tool for discriminating between the three isochromic, 380 nm absorbing intermediates involved in rhodopsin activation and for gaining insight into how their structures differ.

pH dependence of photolysis intermediates in the photoactivation of rhodopsin mutant E113Q.

Glutamic acid at position 113 in bovine rhodopsin ionizes to form the counterion to the protonated Schiff base (PSB), which links the 11-cis-retinylidene chromophore to opsin. Photoactivation of rhodopsin requires both Schiff base deprotonation and neutralization of Glu-113. To better understand the role of electrostatic interactions in receptor photoactivation, absorbance difference spectra were collected at time delays from 30 ns to 690 ms after photolysis of rhodopsin mutant E113Q solubilized in dodecyl maltoside at different pH values at 20 degrees C. The PSB form (pH 5. 5, lambda(max) = 496 nm) and the unprotonated Schiff base form (pH 8. 2, lambda(max) = 384 nm) of E113Q rhodopsin were excited using 477 nm or 355 nm light, respectively. Early photointermediates of both forms of E113Q were qualitatively similar to those of wild-type rhodopsin. In particular, early photoproducts with spectral shifts to longer wavelengths analogous to wild-type bathorhodopsin were seen. In the case of the basic form of E113Q, the absorption maximum of this intermediate was at 408 nm. These results suggest that steric interaction between the retinylidene chromophore and opsin, rather than charge separation, plays the dominant role in energy storage in bathorhodopsin. After lumirhodopsin, instead of deprotonating to form metarhodopsin I(380) on the submillisecond time scale as is the case for wild type, the acidic form of E113Q produced metarhodopsin I(480), which decayed very slowly (exponential lifetime = 12 ms). These results show that Glu-113 must be present for efficient deprotonation of the Schiff base and rapid visual transduction in vertebrate visual pigments.

Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: a flow-flash investigation using optical multichannel detection.

Proton and electron transfer events during the reaction of solubilized fully reduced bovine heart cytochrome c oxidase with molecular oxygen were investigated using the flow-flash technique. Time-resolved spectral changes resulting from ligand binding and electron transfer events were detected simultaneously with pH changes in the bulk. The kinetics and spectral changes in the visible region (450-750 nm) were probed by optical multichannel detection, allowing high spectral resolution on time scales from 50 ns to 50 ms. Experiments were carried out in the presence and absence of pH-sensitive dyes (carboxyfluorescein at pH 6.5, phenol red at pH 7.5, and m-cresol purple at pH 8.5) which permitted separation of spectral changes due to proton transfer from those caused by ligand binding and electron transfer. The transient spectra recorded in the absence of dye were analyzed by singular-value decomposition and multiexponential fitting. Five apparent lifetimes (0.93 microseconds, 10 microseconds, 36 microseconds, 90 microseconds, and 1.3 ms at pH 7.5) could consistently be distinguished and provided a basis for a reaction mechanism consistent with our most recent kinetic model [Sucheta, A., Szundi, I., and Einarsdóttir, O. (1999) Biochemistry 37, 17905-17914]. The dye response indicated that proton uptake occurred concurrently with the two slowest electron transfer steps, in agreement with previous results based on single-wavelength detection [Hallén, S., and Nilsson, T. (1992) Biochemistry 31, 11853-11859]. The stoichiometry of the proton uptake reactions was approximately 1.3 +/- 0.3, 1.4 +/- 0.3, and 1.6 +/- 0.5 protons per enzyme at pH 6.5, 7.5, and 8.5, respectively. The electron transfer between heme a and CuA was limited by proton uptake on a 90 microseconds time scale. We have established the lower limit of the true rate constant for the electron transfer between CuA and heme a to be approximately 2 x 10(5) s-1.

The effects of octanol on the late photointermediates of rhodopsin.

Membrane suspensions of unperturbed rhodopsin and rhodopsin perturbed with 2.5 mM octanol were photolyzed with 477 nm laser pulses at 20 degrees C and 35 degrees C. Changes in absorbance were monitored at times ranging from 1 microsecond to 80 ms after excitation. The data were analyzed using singular value decomposition, global exponential fitting and kinetic modeling. A recently proposed model involving the photointermediate Meta-I380 (T. E. Thorgeirsson, J. W. Lewis, S. E. Wallace-Williams, and D. S. Kliger, Biochemistry 32, 13861-13872, 1993) fits data for samples with and without octanol. Comparison of the microscopic rates shows this alcohol accelerates the formation of Meta-II via Meta-I380. Activation and equilibrium thermodynamic parameters obtained from Arrhenius plots suggest that octanol reduces the entropy increase in forming both Meta-I380 and Meta-II. It also lowers the enthalpy of Meta-I380 relative to Lumi and of Meta-II relative to Meta-I480. To help determine whether octanol affects the protein directly or indirectly through the lipid bilayer, similar experiments were conducted using rhodopsin solubilized in 0.13% dodecyl maltoside with and without octanol. Spectral shifts in the presence of octanol suggest that a direct protein interaction exists in addition to previously reported effects dependent on membrane free volume.

Proton transfer reactions linked to rhodopsin activation.

Purified bovine rhodopsin solubilized in dodecyl maltoside was photolyzed at 20 degreesC with 477 nm light, and difference spectra were collected at time delays ranging from 10 micros to 10 ms after photolysis. Bromocresol purple was added to the samples to detect pH changes in the aqueous environment due to changes in the protonation state of rhodopsin. The data were analyzed using singular value decomposition and global exponential fitting, which revealed three exponential processes indicating the presence of at least four intermediates. Spectral changes of the indicator dye were separated from those of rhodopsin, and proton release and uptake rates were analyzed within the framework of rhodopsin photoreaction kinetics. Proton release occurred during Lumi decay to Meta-I380 followed by uptake upon Meta-I380 decay and by a more significant proton uptake with the time course of Meta-I480 decay. On the basis of the estimated number of protons released and taken up in each step of the rhodopsin photoreaction, we concluded that two forms of Meta-II are present. The two forms of Meta-II, Meta-IIa' and Meta-IIb, differ in protonation state from one another as do both from the earlier, 380 nm absorbing form, Meta-I380.

Effects of pH on rhodopsin photointermediates from lumirhodopsin to metarhodopsin II.

Time-resolved absorption difference spectra of membrane suspensions of bovine rhodopsin at pH 5, 6, 7, 8, 9, and 10 were collected in the time range from 1 micro s to 200 ms after laser photolysis with 7-ns pulses of 477-nm light. The data were analyzed using singular value decomposition (SVD) and global exponential fitting. At pH 7 the data agree well with previously obtained data (Thorgeirsson et al. (1993) Biochemistry 32, 13861-13872) with fits improved at all pH's by inclusion of a small component due to an absorbance change caused by rotational diffusion which is detectable even at magic angle polarization. A "square scheme" suggested to best explain the previous data, which involves two branches following decay of the lumi intermediate with pathways (1) lumi --> MI480 right harpoon over left harpoon MII and (2) lumi right harpoon over left harpoon MI380 --> MII, could be confirmed throughout the entire pH range. However, to account for the increased rate of the MII --> MI480 reaction in path 1 for rising pH values, we propose that the MII in the square scheme consists of deprotonated MII and protonated MIIH+ forms in rapid equilibrium with each other, resulting in an extended square scheme and increasing the number of 380-nm products from two to three. In addition to the kinetic processes described by the extended square scheme, above pH 8 fast ( approximately 10 micro s) and slow ( approximately 50 ms) components were found. The fast component was assigned to the decay of a blue-shifted lumi intermediate, and the slow component, resolvable only at pH 10, was assigned to formation of a 450 nm absorbing photoproduct.

Intermediates in the reaction of fully reduced cytochrome c oxidase with dioxygen.

The reduction of dioxygen to water by cytochrome c oxidase was monitored in the Soret region following photolysis of the fully reduced CO complex. Time-resolved optical absorption difference spectra collected between 373 and 521 nm were measured at delay times from 50 ns to 50 ms and analyzed using singular value decomposition and multiexponential fitting. Five processes were resolved with apparent lifetimes of 0.9 micros, 8 micros, 36 micros, 103 micros, and 1.2 ms. A mechanism is proposed and spectra of intermediates are extracted and compared to model spectra of the postulated intermediates. The model builds on an earlier mechanism that used data only from the visible region (Sucheta et al. (1997) Biochemistry 36, 554-565) and provides a more complete mechanism that fits results from both spectral regions. Intermediate 3, the ferrous-oxy complex (compound A) decays into a 607 nm species, generally referred to as P, which is converted to a 580 nm ferryl form (Fo) on a significantly faster time scale. The equilibrium constant between P and Fo is 1. We propose that the structure of P is a3(4+)=O CuB2+-OH- with an oxidizing equivalent residing on tyrosine 244, located close to the binuclear center. Upon conversion of P to Fo, cytochrome a donates an electron to the tyrosine radical, forming tyrosinate. Subsequently a proton is taken up by tyrosinate, forming F(I) [a3(4+)=O CuB2+-OH- a3+ CuA+]. This is followed by rapid electron transfer from CuA to cytochrome a to produce F(II) [a3(4+)=O CuB2+-OH- a2+ CuA2+].

Properties of early photolysis intermediates of rhodopsin are affected by glycine 121 and phenylalanine 261.

Glycine 121 in transmembrane (TM) helix 3 and phenylalanine 261 in TM helix 6 of bovine rhodopsin have been shown to be critical residues for creating an appropriate chromophore binding pocket for 11-cis-retinal [Han, M., Lin, S. W., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32330-32336; Han, M., Lin, S. W., Minkova, M., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32337-32342]. To further explore structure-function relationships in the vicinity of receptor helices 3 and 6, time-resolved absorption difference spectra of rhodopsin mutants G121A, G121V, and G121L/F261A were obtained at 20 degrees C. Data were collected from 30 ns to 690 ms after laser photolysis with 7 ns pulses (lambdamax = 477 nm) and analyzed using a global exponential fitting procedure after singular value decomposition (SVD). For each mutant, the decay of its bathorhodopsin photoproduct (batho) into an equilibrium with its blue-shifted intermediate (bsi) was too fast to resolve (<20 ns). The reaction scheme found for the mutants G121A and G121L/F261A was batho/bsi --> lumirhodopsin (lumi) --> metarhodopsin I (MI) --> metarhodopsin II (MII). For G121V, an additional early 380 nm absorber, with a back-reaction to lumi, had to be included in the above scheme. For the three Gly121 mutants, the main pathway to reach the active MII state is via lumi and MI. This is in contrast to rhodopsin where the main pathway in detergent samples is via lumi and an early 380 nm absorber, MI380. From the accelerated batho decay present in all three mutants, we conclude that Gly121 is likely to participate in the earliest chromophore-protein interactions. In addition, bsi decay is further accelerated in mutant G121L/F261A, suggesting that Phe261 is an essential determinant of the protein processes involved in bsi decay.

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis.

Rhodopsin is a prototypical G protein-coupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin's transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.

Deriving reaction mechanisms from kinetic spectroscopy. Application to late rhodopsin intermediates.

A general algebraic approach to the kinetic analysis of time-dependent absorption data is presented that allows the calculation of possible kinetic schemes. The kinetic matrices of all possible reaction mechanisms are calculated from experimental eigenvalues and eigenvectors derived from the decay constants and amplitude spectra (b-spectra) of the global exponential fit to the time-dependence of the absorption data. The eigenvalues are directly related to the decay constants, and the eigenvectors are obtained by decomposing the b-spectra into spectral components representing the intermediates. The analysis method is applied to the late intermediates (lumi, meta I, meta I-380, and meta II) of the rhodopsin photoreaction. The b-spectra are decomposed into lumi, meta I, meta-380, and rhodopsin spectra. The meta-380 component is partitioned into isospectral meta I-380 and meta II components based on physical criteria. The calculated kinetic matrices yield a number of reaction mechanisms (linear scheme with back reactions, branched schemes with equilibrium steps, and a variety of square models) consistent with the photolysis data at 25 degrees C. The problems associated with isospectral intermediates (meta I-380 and meta II) are treated successfully with this method.

Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants.

Time-resolved absorption difference spectra of COS-cell expressed rhodopsin and rhodopsin mutants (E113D, E113A/A117E, and G90D), solubilized in detergent, were collected from 20 ns to 510 ms after laser photolysis with 7 ns pulses (lambda(max) = 477 nm). The data were analyzed using a global exponential fitting procedure following singular value decomposition (SVD). Over the entire time range excellent agreement was achieved between results for COS-cell and rod outer segment rhodopsin both in kinetics and in the lambda(max) values of the intermediates. The Schiff base counterion mutant E113D showed strong similarities to rhodopsin up to lumi, following the established scheme: batho <==> bsi --> lumi. Including late delay times (past 1 micros), the mutant E113D lumi decayed to metarhodopsin II (MII), showing that the detergent strongly favors MII over metarhodopsin I (MI). However, a back-reaction from MII to lumi was observed that was not seen for rhodopsin. The kinetic schemes for the mutants E113A/A117E and G90D were significantly different from that of rhodopsin. In both mutants batho decay into an equilibrium with bsi was too fast to resolve (<20 ns). The batho/bsi mixtures decayed with the following reaction scheme: batho/bsi <==> lumi <==> MI-like <==> MII-like. However, the back-reaction from MI-like to lumi was not seen in G90D. MI-like spectral intermediates absorbing around 460 nm appeared in both mutants. They have been shown to be the transducin-activating species (R*). These data, interpreted in the context of previous NMR, FTIR, and Raman data, are consistent with a picture in which the kinetics of batho decay is dependent on a protein-induced perturbation near C12-C13 of the retinal chromophore. The lambda(max) values of the bsi and lumi intermediates in the mutant pigments are interpreted in terms of movement of the Schiff base relative to its counterion.