Improved system for heterologous expression of cytochrome c mutants in Escherichia coli.

We improved an already existing cytochrome c expression system to a reliable, tightly controllable one to achieve a higher expression yield for single cysteine mutants of horse cytochrome c. The protein is heterologously overexpressed in E. coli together with the maturation coordinating enzyme heme lyase from yeast. Various plasmid constructs and host strains were tested for protein expression yield and routinely around 35 mg/L yield was achieved, which is a good result for a post-translationally modified enzyme. The purpose of producing cysteine mutants is to position accessible cysteine residues on the surface of cytochrome c which can be labeled with a photoactive redox dye, 8-thiouredopyrene-1,3,6-trisulfonate, TUPS. TUPS labeled proteins have been used for intramolecular and intermolecular electron transfer measurements. Here, we initiate the photoreduction of cytochrome c oxidase, the natural electron acceptor partner of cytochrome c by an appropriate cytochrome c mutant labeled with TUPS. The electron transfer from cytochrome c to the first cytochrome oxidase redox cofactor, copper A, is shown to be very fast.

Dissecting the photocycle of the bacteriorhodopsin E204Q mutant from kinetic multichannel difference spectra. Extension of the method of singular value decomposition with self-modeling to five components.

Kinetic multichannel difference spectroscopy in the visible spectral range of the Glu204 --> Gln(E204Q) site-directed mutant of bacteriorhodopsin revealed five spectrally distinct metastable intermediates, as for the wild type. Due to the perturbation of the extracellular proton release cluster, the late O intermediate accumulates in much higher amounts in this mutant, and the photocycle is not complicated by the pH-dependent branching observed in the wild type protein. This mutant is therefore more amenable than the wild type to the determination of the intermediate spectra with the method of singular value decomposition with self-modeling, developed recently for three components (Zimányi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4408-4413, 4414-4419). The method provides the most reliable spectra so far, defining the time evolution of the intermediates essential to the determination of the reaction scheme that describes the photocycle. The analysis confirms published results on this mutant by and large, but revises the locations of the L intermediates in the photocycle. In addition, it allows identification of the pH-dependent transitions of the photocycle, and offers an alternative mechanism for the pH dependence of the yield and kinetics of the late O intermediate.

Singular value decomposition with self-modeling applied to determine bacteriorhodopsin intermediate spectra: analysis of simulated data.

An a priori model-independent method for the determination of accurate spectra of photocycle intermediates is developed. The method, singular value decomposition with self-modeling (SVD-SM), is tested on simulated difference spectra designed to mimic the photocycle of the Asp-96 --> Asn mutant of bacteriorhodopsin. Stoichiometric constraints, valid until the onset of the recovery of bleached bacteriorhodopsin at the end of the photocycle, guide the self-modeling procedure. The difference spectra of the intermediates are determined in eigenvector space by confining the search for their coordinates to a stoichiometric plane. In the absence of random noise, SVD-SM recovers the intermediate spectra and their time evolution nearly exactly. The recovery of input spectra and kinetics is excellent although somewhat less exact when realistic random noise is included in the input spectra. The difference between recovered and input kinetics is now visually discernible, but the same reaction scheme with nearly identical rate constants to those assumed in the simulation fits the output kinetics well. SVD-SM relegates the selection of a photocycle model to the late stage of the analysis. It thus avoids derivation of erroneous model-specific spectra that result from global model-fitting approaches that assume a model at the outset.

Intermediate spectra and photocycle kinetics of the Asp96 --> asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling.

Singular value decomposition with self-modeling is applied to resolve the intermediate spectra and kinetics of the Asp96 --> Asn mutant bacteriorhodopsin. The search for the difference spectra of the intermediates is performed in eigenvector space on the stoichiometric plane. The analysis of data at pH values ranging from 4 to 8 and temperatures between 5 and 25 degrees C reveals significant, early partial recovery of the initial state after photoexcitation. The derived spectra are not biased by assumed photocycles. The intermediate spectra derived in the initial step differ from spectra determined in prior analyses, which results in intermediate concentrations with improved stoichiometric properties. Increasingly more accurate photocycles follow with increasing assumed complexity, of which parallel models are favored, consistent with recent, independent experimental evidence.

Interpretation of the spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle.

We have recently introduced a method, made possible by an improved orienting technique using a combination of electric and magnetic fields, that allows the three-dimensional detection of the intramolecular charge displacements during the photocycle of bacteriorhodopsin. This method generates electric asymmetry, a prerequisite for the detection of electric signal on the macroscopic sample, in all three spatial dimensions. Purple membrane fragments containing bacteriorhodopsin were oriented so that their permanent electric dipole moment vectors were perpendicular to the membrane plane and pointed in the same direction. The resulting cylindrical symmetry was broken by photoselection, i. e., by flash excitation with low intensity linearly polarized light. From the measured electric signals, the three-dimensional motion of the electric charge center in the bacteriorhodopsin molecules was calculated for the first 400 microseconds. Simultaneous absorption kinetic recording provided the time-dependent concentrations of the intermediates. Combining the two sets of data, we determined the discrete dipole moments of intermediates up to M. When compared with the results of current molecular dynamics calculations, the data provided a decisive experimental test for selecting the optimal theoretical model for the proton transport and should eventually lead to a full description of the mechanism of the bacteriorhodopsin proton pump.

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin.

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Photocycle of halorhodopsin from Halobacterium salinarium.

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.

Testing BR photocycle kinetics.

An improved K absorption spectrum in the visible is obtained from previous photocycle data for the D96N mutant of bacteriorhodopsin, and the previously obtained M absorption spectrum in the visible and the fraction cycling are confirmed at 25 degrees C. Data at lower temperatures are consistent with negligible temperature dependence in the spectra from 5 degrees C to 25 degrees C. Detailed analysis strongly indicates that there are two intermediates in addition to the first intermediate K and the last intermediate M. Assuming two of the intermediates have the same spectrum and using the L spectrum obtained previously, the best kinetic model with four intermediates that fits the time course of the intermediates is rather unusual, with two L's on a cul-de-sac. However, a previously proposed, more conventional model with five intermediates, including two L's with the same spectra and two M's with the same spectra, also fits the time course of the intermediates nearly as well. A new criterion that tests an individual proposed spectrum against data is also proposed.

Thermal equilibration between the M and N intermediates in the photocycle of bacteriorhodopsin.

The stages in the photocycle of bacteriorhodopsin (BR) involving the M and N intermediates are investigated using a double pulse excitation method. A first (cycling) pulse at 532 nm is followed, with an appropriate time delay, by a second pulse (337, 406, 446, or 470 nm) which induces the M-->BR back-photoreaction. After depletion by the second pulse a repopulation of M in the millisecond range is observed which is interpreted in terms of a thermal N-->M relaxation. It is thus concluded that a (thermal) M<-->N equilibrium accounts for the biphasic decay of M in the BR photocycle. Other models for this stage of the light-driven proton-pump are therefore unnecessary.

Pathway of proton uptake in the bacteriorhodopsin photocycle.

The time courses of chromophore reactions and proton uptake in the second half of the photocycle of the proton pump bacteriorhodopsin (BR) were examined. At pH > 8.5, the kinetics are simplified by the fact that only the M and N intermediates accumulate. The relaxation kinetics after perturbation of M with a second, blue flash confirm that M<==>N equilibration is the only significant cause of the biphasic M decay. With this feature, the analysis of time-resolved difference spectra yields a scheme which contains two sequential N states connected by a nearly unidirectional reaction. The proton uptake from the bulk, as measured with the pH-indicator dye pyranine, occurs during the decay of the first N rather than the recovery of BR. The results thus suggest the model M2(-1)<==>N(-1) + H+ (from the bulk)<==>N(0)-->BR, where the superscripts indicate the protonation state of the protein relative to BR. M2(-1)-->N(-1) is reprotonation of the Schiff base from D96, N(-1) + H+ (from the bulk)-->N(0) is uptake of proton from the cytoplasmic side, and N(0)-->BR represents 13-cis to all-trans reisomerization of the retinal and other relaxations which regenerate the initial state. R227, a residue near D96, affects the deprotonation of D96 more than the subsequent proton uptake. According to the changed [M2(-1)]/[N(-1)] equilibrium in the R227Q protein, interaction between R227 and D96 is responsible for about 1 pH unit of the decrease in the pKa of D96 during the reprotonation of the Schiff base. According to the pH dependencies of the rate constants in the N(-1)<==>N(0) equilibrium in wild-type and R227Q, interaction with R227 lowers the pKa for proton uptake from the bulk by 0.5 pH unit, to 11. We conclude from the proton uptake kinetics that at physiological pH free energy is converted to proton electrochemical potential in this pump not only as protons are released on the extracellular side [Zimányi, L., Váró. G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] but also as protons are taken up on the cytoplasmic side.

Photoreaction of the N intermediate of bacteriorhodopsin, and its relationship to the decay kinetics of the M intermediate.

Because the M photointermediate of recombinant T46V bacteriorhodopsin decays more rapidly and the N intermediate more slowly than in wild-type, the photoreaction of N could be examined without interference from M. We found that between pH 6 and 9 the photoproducts of N included both earlier suggested M-like intermediate and red-shifted R state. However, when the photoexcitation of N was at wavelengths below 500 nm the amount of M-like product decreased with increasing pH, and at pH 9 virtually only R was produced. In the dark, T46V contains an N-like conformer, in increasing amounts with increasing pH like wild-type but in 4-5 times greater concentrations. The photoreaction of this thermally produced state is much like that of the N intermediate. It is associated with the appearance of a slowly decaying M, but we calculate that under most conditions used to follow M in the wild-type photocycle the amount of N-like conformer, and therefore the amplitude of this slow component, will not be significant. The results confirm the suggestion [Fukuda & Kouyama (1992) Biochemistry 31, 11740-11747] that an M-like state is included among the photoproducts of N, but at the same time provide support to photocycle models in which the slow component of the biphasic M decay is attributed not to this secondary photoreaction or to a separate photocycle originating from a heterogeneous initial state, but to thermal equilibration between M and N in a single photocycle.

Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin. The simpler case of the recombinant D96N protein.

The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of their interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Váró, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BRhv-->K<==>L<==>M1-->M2--> BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3 x 3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25 degrees C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein.

The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements.

The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

A residue substitution near the beta-ionone ring of the retinal affects the M substates of bacteriorhodopsin.

The switch in the bacteriorhodopsin photocycle, which reorients access of the retinal Schiff base from the extracellular to the cytoplasmic side, was suggested to be an M1----M2 reaction (Váró and Lanyi. 1991. Biochemistry. 30:5008-5015, 5016-5022). Thus, in this light-driven proton pump it is the interconversion of proposed M substates that gives direction to the transport. We find that in monomeric, although not purple membrane-lattice immobilized, D115N bacteriorhodopsin, the absorption maximum of M changes during the photocycle: in the time domain between its rise and decay it shifts 15 nm to the blue relative to the spectrum at earlier times. This large shift strongly supports the existence of two M substates. Since D115 is located near the beta-ionone ring of the retinal, the result raises questions about the possible involvement of the retinal chain or protein residues as far away as 10 A from the Schiff base in the mechanism of the switching reaction.

Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism.

The light-driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison.

Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis.

Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for halorhodopsin. In buffer containing nitrate, halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two pigments. At the extracellular end of the presumed helix C, however, an arginine residue is found in halorhodopsin, but not in pharaonis halorhodopsin, and an arginine-rich segment between the presumed helices A and B in halorhodopsin is replaced by a less positively charged sequence in pharaonis halorhodopsin (Lanyi, J. K., Duschl, A., Hatfield, G. W., May, K., and Oesterhelt, D. (1990) J. Biol. Chem. 265, 1253-1260). One or both of these alterations may explain the difference in the anion selectivity of the two proteins.

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 1. Comparison of the photocycles of bacteriorhodopsin and halorhodopsin.

We used a gated optical multichannel analyzer to measure transient flash-induced absorption changes in bacteriorhodopsin (BR) and halorhodopsin (HR) and developed criteria for calculating the absorption spectra of the photocycle intermediates and the kinetics of their rise and decay. The results for BR agree with data reported by a large number of other authors. The results for HR in the presence of chloride are consistent with earlier data and reveal an additional intermediate, not previously seen, in the submicrosecond time scale. Although an M412-like intermediate is not in the HR photocycle, a one-by-one comparison of the rest of the intermediates observed for BR and HR indicates a striking similarity between the photocycles of the two bacterial rhodopsins. This was previously not apparent, perhaps because the experimental approaches to the spectroscopy of the two pigments were different and the data were thus more fragmented.

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 2. Effects of anions on the halorhodopsin photocycle.

We find that the photocycle of halorhodopsin (HR) in the presence of nitrate (but not chloride) consists of two parallel series of reactions. The first is essentially the same as that which occurs in the presence of chloride: HRhv----HRK----HRKL----HRL----HRO----HR. The second photocycle, however, which we describe as HRhv----HR'K----HRKO----HRO----HR, seems characteristic of what one would observe in the absence of chloride. Absorption spectra are calculated for all species but HRK and HR'K, which occur at shorter times (less than 60 ns) than we can resolve. At nitrate concentrations between 0.1 and 1 M, the proportion of HR which enters the first kind of photocycle increases in such a way as to suggest that nitrate can substitute for chloride, but much less effectively. At lower anion concentrations, the two photocycles are independent of one another, but at higher concentrations, they interact; i.e., the reaction HRKO----HRO----HRL can be observed. Thus, HRO must be common to the two photocycles. Kinetic fitting of the time dependence of HRL and HRO at different chloride concentrations provides evidence for the participation of chloride in the interconversion of HRL and HRO. The results are consistent with a model in which the photoreaction is influenced by the binding of an anion (either chloride or nitrate) to site II in HR: when an anion is bound, the HRK-initiated HRL-type photocycle is observed, but when the site is not occupied, the HR'K-initiated HRO-type photocycle is seen.

Low-temperature photoreactions of halorhodopsin. 1. Detection of conformational substates of the chromoprotein.

Absorption spectra of halorhodopsin (HR), a retinal protein in the halobacterial membrane, and its photostationary states were determined at 80 K. The absorption lines appear to narrow upon cooling, thereby revealing complex spectral fine structure of the main absorption band in the visible region, characteristic of conformational substates of HR. Illumination causes (1) the redistribution of these substrates and consequent changing of the fine structure ("hole-burning") and (2) the appearance of a hypsoproduct of undefined nature, in addition to the previously described bathoproduct HR600. Bacteriorhodopsin, a related retinal pigment, gives rise only to the bathointermediate (i.e., K590) under these conditions. After warming of illuminated HR to 110 K, and recooling to 80 K, relaxation of the illumination-induced change in spectral fine structure, and decay of the hypsoproduct but not the bathoproduct, was observed. The results are explained with a model in which one ensemble of HR conformational substates at 80 K is converted to another in a photoequilibrium via the excited state, which also produces the batho- and hypsoproducts. The original ensemble can be regained through thermal pathways at a somewhat higher temperature, and only the bathoproduct will decay thermally into the next intermediate of the HR photocycle.