T.E. Meyer

New Photocycle Intermediates in the Photoactive Yellow Protein from Ectothiorhodospira halophila: Picosecond Transient Absorption Spectroscopy

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.

Photoactive yellow protein from the purple phototrophic bacterium, Ectothiorhodospira halophila. Quantum yield of photobleaching and effects of temperature, alcohols, glycerol, and sucrose on kinetics of photobleaching and recovery

A water-soluble yellow protein from E. halophila was previously shown to be photoactive (Meyer, T. E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423). Pulsed laser excitation in the protein visible absorption band (maximum at 445 nm) causes a rapid bleach of color (k = 7.5 x 10(3) s-1) followed by a slower dark recovery (k = 2.6 s-1). This is analogous to the photocycle of sensory rhodopsin II from Halobacterium (which also has k = 2.6 s-1 for recovery). We have now determined the quantum yield of the photobleaching process to be 0.64, which is comparable with that of bacteriorhodopsin (0.25), and is thus large enough to be biologically significant. Although the photoreactions of yellow protein were previously shown to be relatively insensitive to pH, ionic strength and the osmoregulator betaine, the present experiments demonstrate that temperature, glycerol, sucrose, and various alcohol-water mixtures strongly influence the kinetics of photobleaching and recovery. The effect of temperature follows normal Arrhenius behavior for the bleach reaction (Ea = 15.5 kcal/mol). The rate constant for the recovery reaction increases with temperature between 5 degrees C and 35 degrees C, but decreases above 35 degrees C indicating alternate conformations with differing kinetics. There is an order of magnitude decrease in the rate constant for photobleaching in both glycerol and sucrose solutions that can be correlated with the changes in viscosity. We conclude from this that the protein undergoes a conformational change as a consequence of the photoinduced bleach. Recovery kinetics are affected by glycerol and sucrose to a much smaller extent and in a more complicated manner. Aliphatic, monofunctional alcohol-water solutions increase the rate constant for the bleach reaction and decrease the rate constant for the recovery reaction, each by an order of magnitude. These effects do not correlate with dielectric constant, indicating that the photocycle probably does not involve separation or recombination of charge accessible to the protein surface. However, the effects on both bleaching and recovery correlate well with the relative hydrophobicity(as measured by partition coefficients in detergent/water mixtures), in the order of increasing effectiveness:methanol < ethanol < iso-propanol <n-propanol < n-butanol. We conclude that the change in conformation of the protein induced by light exposes a hydrophobic site to the solvent. This suggests the possibility that light exerts its effect in vivo by exposing a region of the protein for binding to a hydrophobic receptor site in the cell, perhaps to a protein analogous to the chemotactic transducers in the cytoplasmic membranes of enteric bacteria.

Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens

Three soluble cytochromes were found in two strains of the halophilic non-sulfur purple bacterium Rhodospirillum salexigens. These are cytochromes C2, C and c-551. Cytochrome C2 was recognized by the presence of positive charge at the site of electron transfer (measured by laser flash photolysis), although the protein has an overall negative charge (pI = 4.7). Cytochrome C2 has a high redox potential (300 mV) and is monomeric (13 kDa). Cytochrome c was recognized from its characteristic absorption spectrum. It has a redox potential of 95 mV, an isoelectric point of 4.3, and is isolated as a dimer (33 kDa) of identical subunits (14 kDa), a property which is typical of this family of proteins. R. salexigens cytochrome c-551 has an absorption spectrum similar to the low redox potential Rb. sphaeroides cytochrome c-551.5. It also has a low redox potential (-170 mV), is very acidic (pI = 4.5), and is monomeric (9 kDa), apparently containing 1 heme per protein. The existence of abundant membrane-bound cytochromes c-558 and c-551 which are approximately half reduced by ascorbate and completely reduced by dithionite suggests the presence of a tetraheme reaction center cytochrome in R. salexigens, although reaction centers purified in a previous study (Wacker et al., Biochim. Biophys. Acta (1988) 933, 299-305) did not contain a cytochrome. The most interesting observation is that R. salexigens contains a photoactive yellow protein (PYP), previously observed only in the extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila. The R. salexigens PYP appears to be slightly larger than that of Ec. halophila (16 kDa vs. 14 kDa). Otherwise, these two yellow proteins have similar absorption spectra, chromatographic properties and kinetics of photobleaching and recovery.

Use of laser flash photolysis time-resolved spectrophotometry to investigate interprotein and intraprotein electron transfer mechanisms

A description is given of the methodology developed in our laboratory for the application of laser flash photolysis to the elucidation of the kinetics and mechanism of electron transfer processes which occur intermolecularly between two protein molecules within a collisional complex, or intramolecularly between two redox centers within a single multisubunit or multidomain protein. This involves the use of flavin analogs, excited to their lowest triplet state by a laser flash, to initiate electron transfer, either by oxidation of a sacrificial donor followed by redox protein reduction via the flavin semiquinone, or by direct oxidation of a reduced redox protein by the flavin triplet. Time-resolved spectrophotometry is used to follow the course of the sequence of electron transfer events initiated by the laser flash. The application of this methodology to the following systems is described: cytochrome c/cytochrome c peroxidase; ferredoxin/ferredoxin NADP+ reductase; cytochrome c/plastocyanin; flavocytochrome b2; and sulfite oxidase.

Oxidative turnover increases the rate constant and extent of intramolecular electron transfer in the multicopper enzymes, ascorbate oxidase and laccase

Using laser flash photolysis of lumiflavin/EDTA solutions containing ascorbate oxidase, we find that the rate constant for intramolecular electron transfer varies from one enzyme preparation to another and is generally a more sensitive measure of the state of the active site than are steady-state assays. Thus, type I copper is initially reduced in a second-order reaction followed by first-order reoxidation by the type II-III trinuclear copper center. The observed rate constant for this intramolecular process in presumably native enzyme is 160 s-1 at pH 7, whereas an enzyme preparation which had less than 20% activity had a rate constant of 2.6 s-1. Other samples of relatively active enzyme showed biphasic intramolecular kinetics intermediate between the above values. The inactive enzyme sample could be reactivated by dialysis against ascorbate or by treatment with ferricyanide, resulting in a corresponding increase in the intramolecular rate constant for type I copper reoxidation to a value comparable to that of native enzyme. Using this same methodology, we have determined that the type I copper in Japanese lacquer tree laccase is reoxidized by the type II-III trinuclear copper center in a first-order (intramolecular) process with rate constants of 1 s-1 at pH 7.0 and 4.9 s-1 at pH 6.0, values which are approximately two orders of magnitude smaller than for ascorbate oxidase. The intramolecular rate constant and enzyme activity for laccase also increased, but only by a factor of 2-6, when the enzyme was treated with ascorbate or ferricyanide, respectively. We further found that intramolecular electron transfer in laccase was completely inhibited by fluoride ion, in contrast to ascorbate oxidase which is unaffected by this ion. These differences in behavior for these two very similar enzymes are rather remarkable, when it is considered that the distance between copper atoms is constrained by the location of the protein-derived copper ligands in the three-dimensional structure, and that the redox potentials of the enzymes are similar. Our results may be interpreted in terms of an interconversion between active and inactive enzyme in which there is a rearrangement of the type II-III trinuclear copper center, resulting in a lowering of the redox potential and a block in electron transfer. Turnover restores the active enzyme conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Unusual high redox potential ferredoxins and soluble cytochromes from the moderately halophilic purple phototrophic bacterium Rhodospirillum salinarum

Abstract Two soluble cytochromes and two high redox potential ferredoxins (HiPIP) were purified from extracts of the halophilic non-sulfur purple phototrophic bacterium Rhodospirillum salinarum. All four proteins are highly acidic, as are those from other halophilic phototrophs. Cytochrome c′ is otherwise like those of other species. Cytochrome c-551 has a low redox potential (−143 mV), and appears to be monomeric (12 kDa). We did not find a protein similar to the cytochrome c2 in most species of non-sulfur purple bacteria. The two HiPIP isozymes in R. salinarum differ in native molecular weight (iso-1, 10000 and iso-2, 45000), although iso-2 HiPIP may be a tetramer (subunit size 11 kDa). The redox potential of Iso-1 HiPIP is 265 mV, but iso-2 HiPIP is labile to ferricyanide and other oxidants tested, thus the redox potential was not measured. This is to our knowledge the first report of either an aggregated or a labile HiPIP. Abundant membrane bound c-type cytochromes were observed in R. salinarum by difference spectroscopy. About one-half of the heme (alpha peak maximum 553 nm) could be reduced by ascorbate, whereas the remainder of the heme was reduced by dithionite (552 nm maximum). These observations suggest the presence in R. salinarum of a tetraheme reaction center cytochrome which overshadows the cytochrome bc1 complex.

Photobleaching of the photoactive yellow protein from Ectothiorhodospira halophila promotes binding to lipid bilayers: evidence from surface plasmon resonance spectroscopy

The photoactive yellow protein (PYP) from the phototrophic bacterium Ectothiorhodospira halophila is a small, soluble protein that undergoes reversible photobleaching upon blue light irradiation and may function to mediate the negative phototactic response. Based on previous studies of the effects of solvent viscosity and of aliphatic alcohols on PYP photokinetics, we proposed that photobleaching is concomitant with a protein conformational change that exposes a hydrophobic region on the protein surface. In the present investigation, we have used surface plasmon resonance (SPR) spectroscopy to characterize the binding of PYP to lipid bilayers deposited on a thin silver film. SPR spectra demonstrate that the net negatively charged PYP molecule can bind in a saturable manner to electrically neutral, net positively, and net negatively charged bilayers. Illumination with either blue or white light of a PYP solution, which is in contact with the bilayer, at concentrations below saturation results in an increase in the extent of binding, consistent with exposure of a high affinity hydrophobic surface in the photobleached state, a property that may contribute to its biological function. A value for the thickness of the bound PYP layer (23 A), obtained from theoretical fits to the SPR spectra, is consistent with the structure of the protein determined by x-ray crystallography and indicates that the molecule binds with its long axis parallel to the membrane surface.

Protein interaction sites obtained via sequence homology. The site of complexation of electron transfer partners of cytochrome c revealed by mapping amino acid substitutions onto three-dimensional protein surfaces

Amino acid substitutions in all but the most divergent of cytochromes c have been categorized as being conservative or radical and mapped onto the three-dimensional structure of yeast cytochrome c. Color-coded, space-filling representations reveal a large 24 A diameter surface area which is invariant or conservatively substituted on the front left face of the cytochrome c molecule. Chemical modifications and mutations which inhibit complex formation and electron transfer with reaction partners also map to this surface. In sharp contrast, the back side of the protein is randomly substituted with both conservative and radical replacements. The invariant/conservatively substituted surface on the front of cytochrome c thus defines the site of interaction with redox partners and provides a measure of its dimensions. Further, this analysis strongly suggests that there is only a single site of oxidation and reduction on cytochrome c for all of its physiological reactions. The same analysis applied to bacterial cytochrome c2 shows that its conserved surface is similar in size and location to that of cytochrome c. Analyses of native and model reaction partners of cytochromes c and c2, such as cytochrome b5, plastocyanin, and bacterial photosynthetic reaction centers, also reveal probable active site surfaces for complexation and electron transfer, which are complementary in size to that of the c-type cytochromes. The availability of a three-dimensional structure and of several closely related amino acid sequences for a given functional class of protein is the only limitation on this type of analysis, which can then serve as a basis for designing site-directed mutagenesis experiments.

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.

Evolution of cytochromes and photosynthesis

The following is an outline of the direction of research into the evolutionary origins of photosynthesis as revealed by the study of cytochromes c. Determination of the numbers of kinds of cytochromes, their structures, their functional roles, and their distribution are the principal kinds of data being collected and analyzed. A hypothesis on the origin of photosynthesis is presented.