The plant blue light receptor, phot1, a member of the phototropin family (1), is a plasma membrane-associated flavoprotein that contains two (ϳ110 amino acids) flavinbinding 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. Near-UV blue light regulates a variety of different responses in higher plants. These include phototropism, the inhibition of hypocotyl elongation, the expression of various genes, and stomatal opening. Phot1 (nph1), the recently discovered blue light receptor, is a member of the phototropin receptor family (1). Phot1 is a plasma membrane-associated flavoprotein that functions as the primary photoreceptor mediating phototropic plant movement (2-4). Phot1 has two 12.1-kDa flavin-binding domains, LOV1 and LOV2, within its N-terminal region and a typical serinethreonine protein kinase domain at the C-terminal region. Heterologous expression studies have shown that phot1 binds FMN 1 as a chromophore and undergoes autophosphorylation in response to light treatment. It has therefore been proposed that this receptor functions as a light-activated serine/threonine kinase (4). The isolated LOV domains from oat phot1 expressed in Escherichia coli have been shown to undergo a cyclic photoreaction upon the absorption of light; LOV1 recovers with a half-time of 11.5 s, whereas LOV2 recovers with a half-time of 27 s (5). In addition, the quantum efficiencies for photoproduct (adduct) formation for LOV1 and LOV2 are ϳ0.045 and 0.44, respectively (5). The ground forms of the LOV domains have major absorption peaks at 360 and 450 nm with the 450 peak having vibronic structure at 425 and 475 nm. Upon absorption of light, the chromophore bleaches 2 in the 450 nm region generating a species that absorbs maximally at 390 nm. This intermediate has been assigned as a flavin-cysteinyl adduct between the protein and the C(4a) carbon of the FMN chromophore. This adduct breaks down spontaneously, returning the protein to its ground form. A LOV2 mutant (LOV2C39A) in which the cysteine that forms the adduct has been mutated to alanine does not undergo this photoreaction (5).Recently the crystal structure of the LOV2 domain from the fern Adiantum capillus-veneris phy3 (6) was solved to 2.7-Å resolution (7). Phy3 is a chimeric photoreceptor with homology to phytochrome at its N-terminal end and an almost complete phototropin at its C-terminal end. Its LOV2 domain shares a 70% sequence homology to the oat phot1 LOV2 (6). The structure indicates that the FMN molecule is held noncovalently within...

A cross-linked histidine-phenol compound was synthesized as a chemical analogue of the active site of cytochrome c oxidase. The structure of the cross-linked compound (compound 1) was verified by IR, (1)H and (13)C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pK(a) of the phenolic proton on compound 1 (8.34) was lower than the pK(a) of tyrosine (10.1) or of p-cresol (10.2). This decrease in pK(a) is consistent with the hypothesis that a cross-linked histidine-tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time-resolved optical absorption spectra of compound 1 at room temperature, generated by excitation at 266 nm in the presence and absence of dioxygen, indicated a species with absorption maxima at approximately 330 and approximately 500 nm, which we assign to the phenoxyl radical of compound 1. The electron paramagnetic resonance (EPR) spectra of compound 1, obtained after UV photolysis, confirmed the generation of a paramagnetic species at low temperature. Because the cross-linked compound lacks beta-methylene protons, the EPR line shape was dramatically altered when compared to that of the tyrosyl radical. However, simulation of the EPR line shape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground-state Fourier transform infrared (FT-IR) spectrum of compound 1 showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. We postulate that phenol oxidation produces a small delocalization of spin density onto the imidazole nitrogen of compound 1, which may explain its unique optical spectral properties.

The phototropins are a family of membrane-associated flavoproteins that function as the primary blue light receptors regulating phototropism, chloroplast movements, stomatal opening, and leaf expansion in plants. Phot1, a member of this family, contains two FMN-binding domains, LOV1 and LOV2, within the Nterminal region and a C-terminal serine-threonine protein kinase domain. Light irradiation of oat phot1 LOV2 produces a cysteinyl adduct (Cys-39) at the flavin C(4a) position, which decays thermally back to the dark state. We measured pH and isotope effects on the photocycle. Between pH 3.7 and 9.5, adduct formation showed minimal pH dependence, and adduct decay showed only slight pH dependence, indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly 5-fold slowing of adduct formation in D 2 O relative to H 2 O, indicating that the ratelimiting step involves proton transfer(s). Light-induced changes in the far UV CD spectrum of LOV2 revealed putative protein structural perturbations. The light minus dark CD difference spectrum resembles an inverted ␣-helix spectrum, suggesting that ␣-helicity is reversibly lost upon light irradiation. Decay kinetics for CD spectral changes in the far UV region occur at the same rate as those in the visible region, indicating synchronous relaxation of protein and chromophore structures.The phototropins are a family of blue light receptors that are responsible for phototropism (1, 2) and are involved in lightinduced chloroplast movements (3) as well as blue light-stimulated stomatal opening (4) and leaf expansion in higher plants (5). Phototropin-like proteins have recently been identified in the green alga Chlamydomonas reinhardtii (6, 7) and in bacteria (8). The physiological roles of such proteins in these latter systems are not yet elucidated.The phototropin phot1 (9, 10), which becomes autophosphorylated in response to blue light, is a membrane-associated protein that contains two 12-kDa, FMN-binding LOV (light, oxygen, voltage) (11, 12) domains (LOV1 and LOV2) in its N-terminal region and a typical serine-threonine kinase domain in its C-terminal region (11). LOV domains belong to the PAS domain superfamily, which are found in a variety of sensor proteins in organisms ranging from archaea to eukaryotes (13).Upon light excitation, the isolated LOV2 domain of phot1 undergoes a cyclic photoreaction (14). The photocycle of phot1 LOV2 has been elucidated in part (15). Blue light irradiation excites the FMN chromophore to a triplet state that absorbs maximally around 660 nm (designated LOV2 Despite the above progress made in understanding the phot1 LOV2 photochemistry, specific mechanistic and conformational steps of the photocycle remain to be elucidated. Adduct decay for the LOV2 domain of oat phot1 has been shown to be 3 times slower in D 2 O than in H 2 O (15), indicating that proton transfer reactions, probably involving at least N-5, are rate-limiting components of the back reaction. To date, no comparable information has...

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+].

Sensory rhodopsin I (sR-I) is a phototaxis receptor in halobacteria, which is closely related to the lightdriven proton pump bacteriorhodopsin and the chloride pump halorhodopsin found in the same organisms. The three pigments undergo similar cyclic photoreactions, in spite of their different functions. In intact cells or isolated membranes sR-I is complexed with protein HtrI, the next link in the signal transduction chain, and does not function as an electrogenic ion pump. However, mumination of sR-I in membranes lacking HtrI causes pH changes in the medium, and its photoreaction kinetics become pH-dependent. We show here that in closed vesicles, near neutral pH it functions as an electrogenic proton pump capable of generating at least -80 mV transmembrane potential. The action spectrum shows a maximum 37 um below the 587-nm absorption maximum of the native pigment. This apparent discrepancy occurs because the 587-nm form of HtrI-free sR-I exists in a pH-dependent equilibrium with a 550-nm absorbing species generated through deprotonation of one group with a pK. of 7.2, which we have tentatively identified as Asp-76. We interpret the results in terms of a general model for ion translocation by the bacterial rhodopsins.

Purple membrane (ma.. = 568 nm) can be converted to blue membrane (kma. = 605 nm) by either acid titration or deionization. Partially delipidated purple membrane, containing only 25% of the initial lipid phosphorus, could be converted to a blue form by acid titration but not by deionization. This reversible transition of delipidated mem, brane did not require the presence of other cations, and the pK of the color change that in native membrane under similar conditions is between 3.0 and 4.0 was shifted to 1.4. We conclude that the purple-to-blue transition is controlled by proton concentration only and that, in native membranes, the cations act only by raising the low surface pH generated by the acidic groups of the lipids. The observation that extraction of lipids from deionized native membrane converts its color from blue to purple further confirms this conclusion. The two states of the membrane probably reflect two preferred conformations of bacteriorhodopsin, which are controlled by protonation changes at the surface of the membrane and differ slightly in the spatial distribution of charges around the chromophore.The retinylidene protein bacteriorhodopsin (bR) functions as a light-driven proton pump (1, 2). It is the only protein in the purple membrane (pm) and is arranged in a hexagonal lattice (3, 4). Purple membrane contains a variety of diether lipids, amounting to about 25% by weight, that fill the spaces between bR molecules in the lattice and are all in close contact with the protein (3-6). Most of the lipids are acidic (80%); 70% are phospholipids, mostly the diether analogue of phosphatidylglycerophosphate, and 30% are glycosulfolipids (7,8).Acidification or removal of cations from pm suspensions shifts the absorption maximum of bR from 568 to 605 nm (1, 9-15). The chromophores in these acid or deionized blue membranes are indistinguishable in absorption and resonance Raman spectra (16). The color of bR is apparently controlled by the distribution of charges in the chromophore, specifically protonation and a charge pair or dipole near the P-ionone ring that cause a large red shift of the retinal absorbance; a counterion-i.e., a negatively charged amino acid residue near the protonated Schiff base-stabilizes it with a concomitant reduction of the red shift (17-19). Protonation of this counterion has been suggested as the cause for the purple-to-blue transition. The purple color returns upon further acidification and formation of this "acid purple" chromophore has been attributed to restoration of the negative charge by binding of an anion at the site protonated in the purple-to-blue transition (11) or to protonation of a second negatively charged group (12).The original purple color of the membrane can be restored by addition of salt and alkalinization. Binding of three to five cations to specific sites is generally believed to be required for the color change (13,14,20,21) and the binding sites with the highest affinity may not affect the color change (21). Red shifts of the bR absorption ba...

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba 3 cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O 2 -carrier. A novel doublelaser excitation is introduced in which dioxygen is generated by photolyzing the O 2 -carrier with a 355 nm laser pulse and the fully reduced CO-bound ba 3 simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O 2 binding to heme a 3 at 90 μM O 2 occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 × 10 9 M −1 s −1 for O 2 binding to ba 3 in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O 2 bond cleavage in ba 3 of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O 2 to the active site in ba 3 and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a 3 site in ba 3 , making the CO flow-flash method inapplicable.double-laser technique | T. thermophilus ba3 | oxygen reduction | slow-fast kinetics | O2 channel T he reduction of dioxygen to water in the heme-copper oxidases takes place at the high-spin heme a 3 and Cu B heterodinuclear center (for review, see refs. 1 and 2). The reaction has been extensively investigated in several aa 3 -oxidases by timeresolved spectroscopic techniques in combination with the CO flow-flash technique (1, 2), in which the reaction is initiated by photolyzing CO bound to heme a 3 2þ in the presence of O 2 (3). The O 2 reduction has commonly been interpreted in terms of a unidirectional sequential mechanism (Scheme 1).The O 2 reduction in Thermus thermophilus ba 3 , a B-type oxidase with distant sequence homology to the A-type oxidases (4), has received much less attention (5-7). The enzyme contains the four redox-active metal centers (8-10) and functions as a terminal oxidase for aerobic metabolism under limited oxygen concentration (8-11). It also possesses NO reductase activity (12) suggesting shared evolutionary lineage of O 2 ∕NO reduction in this enzyme. In ba 3 , the thermal dissociation of CO from heme a 3 2þ in the dark is significantly faster (0.8 s −1 ) (5) than in the bovine aa 3 (0.023 s −1 ) (3, 13), and therefore CO flow-flash experiments on ba 3 require fast mixing; such experiments have recently been reported (6, 7). Moreover, the Cu B þ -CO complex formed following CO photolysis from heme a 3 2þ in ba 3 decays with a lifetime of approximately 30 ms (14), a rate much slower than that of O 2 binding to heme ...

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.