Investigations of optical line shapes and kinetic hole burning in myoglobin.

We present the results of an extensive investigation of the optical line shapes of deoxymyoglobin (Mb), the ligand-bound form (MbCO), and the low-temperature photoproduct (Mb*). The thermal properties and the pH dependence of the Soret band and the near infrared band III (approximately 760 nm) are analyzed, taking into account the underlying vibrational properties of the absorption bands. The strong temperature dependence associated with the Soret band of MbCO and band III of Mb indicates significant coupling to low-frequency modes that may not be directly observed in the resonance Raman spectra. On the basis of analogous line-shape studies in a variety of heme systems, we assign the low-frequency coupling in MbCO to torsional motions of the CO molecule. The low-frequency mode coupled to band III (approximately 70 cm-1) is found to lie quite close to the value for the heme-doming motion (approximately 50 cm-1) calculated by using the kinetically determined value of the force constant (17 N/m). Significant inhomogeneous broadening in the Soret region of Mb and Mb* is found to be due to a "nonkinetic" coordinate that we associate with the orientation of the proximal histidine. A "kinetic" coordinate, associated with the equilibrium displacement of the iron atom from the porphyrin plane (a) is found to contribute to the inhomogeneous broadening of both the Soret band and band III. The relaxation of the heme as the system evolves from from Mb* to Mb is followed optically as a function of temperature, and a sharp transition temperature is found at 185 K. The blue shifts of the Soret band and band III as Mb* evolves to Mb are found to be nearly identical (delta v*ABS approximately 140 cm-1) and attributed to changes in the mean value of a between Mb* (a*0) and Mb (a0 = 0.45 A). A simple quadratic model for the coordinate coupling that simultaneously accounts for the observed shift, delta v*ABS, the low-temperature kinetics and the kinetic hole burning predicts a*0 = 0.2 +/- 0.05 A and EA = 16 +/- 2 kJ/mol for the room temperature Arrhenius barrier height at the heme. A simple quantitative method for the analysis of kinetic hole-burning experiments is also developed and applied to recent studies involving quaternary and subunit-specific hemoglobin structures.     

Kinetics and temperature dependence of carboxymyoglobin ligand photodissociation

We have observed the rate of oxymyoglobin (MbO2) photodissociation at room temperature and carboxymyoglobin (MbCO) photodissociation as a function of temperature (260-10 K) by means of picosecond spectroscopy. The Mb + O2 and Mb + CO photodissociated states have also been characterized. Based on the picosecond experimental data, we postulate that the photodissociation of ligated myoglobin is a nonactivitated process, and the mechanism involves either a small enthalpy barrier or none at all.     

Stimulation of bull seminal Ca2+, Mg2+-dependent endonuclease by various DNA-binding proteins

We have measured ΔA transient absorption spectra in the Soret region and kinetics of photodissociation of oxymyoglobin (MbO₂) solutions following excitation by pulses of duration 350 fsec and 10 μJ energy at 307 nm. We observed an instantaneous bleaching of the absorbance at 414 nm and the appearance of a broad, red-shifted absorption band in the 438–470 nm region with a time constant of 250 fsec indicative of the formation of a short-lived deliganded Mb^★ species which relaxes to the stable Mb with a constant of 3.5 psec. Following this early relaxation, changes in absorption kinetics indicate also a geminate recombination process of constant τ = 100 psec. These data demonstrate that the well established low quantum yield (φ = 0.03) of photodissociation in MbO₂ is related both to the relaxation of an excited Mb^★ state and to a fast geminate recombination process.     

Kinetic evidence for three photolyzable taxonomic conformational substates in oxymyoglobin

The kinetics of oxygen geminate binding with the taxonomic substates of MbO2 are reported. The maximum entropy method was used to analyze the rebinding kinetics of MbCO and MbO2 monitored in the Soret. The resulting rate distributions were found to consist of a small number of overlapping bands. A global parametric fit of a series of rate distributions recorded at several temperatures was performed using a Gaussian basis set to resolve the individual enthalpy distributions P(H). This approach was first validated by showing that the well-documented taxonomic substates of MbCO could be recovered. The method was then applied to MbO2. Three taxonomic substates were identified at pH 4.8, whereas only two of them contribute to oxygen geminate rebinding at pH 7.0. These findings show that, similarly to MbCO, MbO2 also exists as three photolyzable and kinetically different taxonomic substates and suggest reconsidering the issue of the photolysis quantum yield of MbO2.     

4-vinyl and 2,4-divinyl deuteration effects on the low frequency resonance Raman bands of myoglobin: correlation with the structure of vinyl group

Resonance Raman spectra of myoglobin (Mb) reconstituted with 4-vinyl and 2,4-divinyl deuterated protoheme IX were studied in several oxidation, spin, and ligation states (deoxyMb, MbO2, MbCO, metMbH2O, and metMbCN-) with special attention to the low frequency vibrations. Frequency shifts observed on deuteration enabled us to assign some Raman bands to vibrations specifically involving the 2- or 4-vinyl group. The most significant deuteration effect was found for a band around 410 cm-1 in the ferrous state, which loses intensity on 4-vinyl deuteration and is ascribed to a porphyrin in-plane vibration strongly coupled with the skeletal bend of the vinyl group at position 4. Such strong coupling implies that the vinyl group lies in the same plane as the pyrrole II ring, as in the crystalline state. Thus, frequency shifts on vinyl deuteration may be useful as a probe of the orientation of the vinyl group. Resonance Raman spectra of Mb coordinated with various sizes of isocyanides are interpreted in terms of vinyl orientational changes induced by ligation.     

Dioxygen replacement reaction in myoglobin

The replacement reaction of myoglobin (Mb), MbCO + O2 leads to MbO2 + CO leads to MbCO + O2, has been studied with flash photolysis in the temperature range from 140 to 320 K and the time range from 2 mus to 200 s. In a fraction of the Mb, the photodissociated CO remains within the protein; rebinding is not affected by the presence of O2 and occurs with rates that are identical with the ones observed earlier in solvents containing only CO. In the remaining fraction CO migrates into the solvent and Mb combines preferentially with oxygen. The rate of the subsequent replacement of O2 by CO permits calculation of the oxygen dissociation rate ko2; ko2 has been determined from 260 to 320 K. The measurements support a multibarrier model.     

Ligand binding to heme proteins: connection between dynamics and function

Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.     

Intermediate states in ligand photodissociation of carboxymyoglobin studied by dispersive X-ray absorption

The ligand photodissociation of sperm whale carboxymyoglobin (MbCO) at low temperature (15 K-100 K) under extended illumination has been studied by X-ray Absorption Near Edge Structure (XANES) spectroscopy using the dispersive technique. XANES simulations through the multiple scattering (MS) approach allow one to interpret the spectroscopic data in structural terms, and to investigate the Fe site structure configurations of the states that follow the CO photodissociation as a function of temperature. The Fe site in the photoproduct is unbound, with an overall structure similar to the deoxy-form (Mb) of the protein. The Fe site structure changes from T < 30 K (Mb^*) to T>50 K (Mb^**), revealing the existence of a slower unbound state Mb^**. A model is proposed which includes the faster state (Mb^*) as a planar porphyrin ring with a displacement of Fe from the heme plane of less than 0.3 Å, and the slower state (Mb**) with a domed heme. Correspondence to: S. Della Longa     

Carboxy Mb at pH 3. Time-resolved resonance Raman study at cryogenic temperatures.

Cryogenic samples of MbCO at pH3 are studied using nanosecond and picosecond time-resolved resonance Raman spectroscopy. It is observed that under excitation conditions sufficient to completely photodissociate MbCO at pH7, the pH3 sample at 10 ns remains substantially unphotolyzed even at 15 K. The similarity in the optical and resonance Raman spectra of MbCO at pH3 with that of pH7 indicates that at pH3 the iron remains six-coordinate and low-spin. The Fe-CO stretch frequency is consistent with a more upright CO orientation. The absence of the v(Fe-His) band in the 30 ps photoproduct Raman spectrum suggests that the Fe-His(F8) bond is broken within 30 ps of photodissociation. Other Raman bands, though, are not consistent with a normal four-coordinate heme for the photoproduct, Mb*. Suggested possible interpretations include a four-coordinate heme highly perturbed by the close lying protonated proximal histidine or a five-coordinate heme with the Fe-His bond significantly weakened. The partial photolysis monitored at 30 ps and 100 K indicates either a significant amount of geminate recombination within 30 ps or low quantum yield or photolysis. The time course for CO recombination is monitored via the Raman spectra from 30 ps to 3 ns at 100 K and 160 K. Of the fraction of protein-ligand pairs that remain photodissociated at 30 ps, 50% recombine by approximately 250 ps at 100 K and 160 K, supporting the flash photolysis rebinding data of Cowen et al. (Cowen, B. R. 1990. Ph. D. thesis. University of Illinois at Urbana-Champaign; Cowen, B. R., D. Braunstein, H. Frauenfelder, P. J. Steinbach, and R. D. Young. 1989. Biophys. J. 55:55a. [Abstr.].) The conclusions from these resonance Raman studies are extended to solution phase studies at ambient temperatures.     

Time-resolved resonance Raman study on ultrafast structural relaxation and vibrational cooling of photodissociated carbonmonoxy myoglobin

A localized small structural change is converted to a higher order conformational change of protein and extends to a mesoscopic scale to induce a physiological function. To understand such features of protein, ultrafast dynamics of myoglobin (Mb) following photolysis of carbon monoxide were investigated. Recent results are summarized here with a stress on structural and vibrational energy relaxation. The core expansion of heme takes place within 2 ps but the out of plane displacement of the heme iron and the accompanying protein conformational change occur in 10 and 100 s of the picosecond regimes, respectively. Unexpectedly, it was found from UV resonance Raman spectra that Trp7 in the N-terminal region and Tyr151 in the C-terminal region undergo appreciable structural changes upon ligand binding-dissociation while Tyr104, Tyr146, and Trp14 do not. Because of the communication between the movements of these surface residues and the heme iron, the rate of spectral change of the iron-histidine (Fe- His) stretching band after CO photodissociation is influenced by the viscosity of solvent. Temporal changes of the anti-Stokes Raman intensity demonstrated immediate generation of vibrationally excited heme upon photodissociation and its decay with a time constant of 1-2 ps.     

Application of molecular dynamics and free energy perturbation methods to metalloporphyrin-ligand systems II: CO and dioxygen binding to myoglobin.

The protein contribution to the relative binding affinity of the ligands CO and O2 toward myoglobin (Mb) has been simulated using free energy perturbation calculations. The tautomers of the His E7 residue are different for the oxymyoglobin (MbO2) and carboxymyoglobin (MbCO) systems. This was modeled by performing two-step calculations that mutate the ligand and mutate the His E7 tautomers in separate steps. Differences in hydrogen bonding to the O2 and CO ligands were incorporated into the model. The O2 complex was calculated to be 2-3 kcal/mol more stable than the corresponding CO complex when compared to the same difference in an isolated heme control. This value agrees well with the experimental value of 2.0 kcal/mol. In qualitative agreement with experiments, the Fe-C-O bond is found to be bent (theta = 159.8 degrees) with a small tilt (theta = 6.2 degrees). The contributions made by each of the 29 residues--within the 9.0-A radius of the iron atom--to the free energy difference are separated into van der Waals and electrostatic contributions; the latter contributions are dominant. Aside from the proximal histidine and the heme group, the residues having the largest difference in free energy in mutating MbO2-->MbCO are His E7, Phe CD1, Phe CD4, Val E11, and Thr E10.     

Rearrangement of the distal pocket accompanying E7 His----Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket

Two-dimensional 1H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO2). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C zeta H proton of the Phe CD4 was found to move toward the iron of the heme by approximately 4 A relative to the position of sperm whale MbCO, requiring minimally a 3-A movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His----Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.     

Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.     

Sequential photoisomerisation dynamics of the push-pull azobenzene Disperse Red 1

The ultrafast dynamics of the push-pull azobenzene Disperse Red 1 following photoexcitation at λ(pump) = 475 nm in solution in 2-fluorotoluene have been probed by broadband transient absorption spectroscopy and fluorescence up-conversion spectroscopy. The measured two-dimensional spectro-temporal absorption map features a remarkable "fast" excited-state absorption (ESA) band at λ ≈ 570 nm appearing directly with the excitation laser pulse and showing a sub-100 fs lifetime with a rapid spectral blue-shift. Moreover, its ultrafast decay is paralleled by rising distinctive ESA at other wavelengths. Global fits to the absorption-time profiles using a consecutive kinetic model yielded three time constants, τ(1) = 0.08 ± 0.03 ps, τ(2) = 0.99 ± 0.02 ps, and τ(3) = 6.0 ± 0.1 ps. Fluorescence-time profiles were biexponential with time constants τ(1)' = 0.12 ± 0.06 ps and τ(2)' = 0.70 ± 0.10 ps, close to the absorption results. Based on the temporal evolution of the transient spectra, especially the "fast" excited-state absorption band at λ ≈ 570 nm, and on the global kinetic analysis of the time profiles, τ(1) is assigned to an ultrafast transformation of the optically excited ππ* state to an intermediate state, which may be the nπ* state, τ(2) to the subsequent isomerisation and radiationless deactivation time to the S(0) electronic ground state, and τ(3) to the eventual vibrational cooling of the internally "hot" S(0) molecules.     

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.     

Fructose-induced modifications of myoglobin: Change of structure from met (Fe3+) to oxy (Fe2+) form

We studied structural modifications of metmyoglobin (Mb) after short-term (6 days) and long-term (30 days) glycation by fructose (fructation). Fructation caused gradual changes in the structure of the protein with respect to increased absorbance at 280 nm, enhanced fluorescence emission (with excitation at 285 nm), increased surface accessible tryptophan residues and reduced α-helix content and change in tertiary structure. However, long-term fructation changed Mb to oxymyoglobin (MbO2), as demonstrated by different spectroscopic (absorption, fluorescence, circular dichroic and electron paramagnetic resonance) studies and trifluoperazine-induced oxygen release experiment. Fructation appeared to modify Arg139 to arg-pyrimidine, which exhibited antioxidative activity and might be involved in the conversion of met (Fe3+) to oxy (Fe2+) form of myoglobin.     

Optical and ESR-spectroscopic study of electronic adducts of oxymyoglobin and oxyhemoglobin

It has been shown that low temperatures (77 degrees K) irradiation of frozen water-glycerol solutions of oxymyoglobin and oxyhemoglobin induces kinetically stabilized nonequilibrium electronic adducts (MbO2-, HbO2-) at the expense of binding of thermolyzed electrons formed during matrix radiolysis to oxygenated hem iron. The absorption spectra of HbO2-and MbO2- have a wide band with the maximum at 545 nm and Soret's band at 421 nm. At 77 K MbO2- gives the ESR spectrum with g beta 1 = 2.203 and g beta 2 = 2.103. Unlike the latter HbO2- ESR spectrum consists of two signals g beta 1 = 2.234, g beta 2 = 2.135 and g alpha 1 = 2.195, g alpha 2 = 2.103. Two signals in HbO2- spectra are shown to be conditioned by electronic adducts of oxygenated alpha- and beta-subunits. The observed effect points to non-equivalency of O2 in alpha- and beta-subunits of oxyhemoglobin. Binding of inositolhexaphopshate to oxyhemoglobin induces changes in the electron structure of HbO2-active centres.     

Rebinding and relaxation in the myoglobin pocket

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.     

Excitation energy pathways in the photosynthetic units of reaction center LM- and H-subunit deletion mutants of <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis>

Light-induced reaction dynamics of isolated photosynthetic membranes obtained from wild-type (WT) and reaction center (RC)-subunit deletion strains SPUHK1 (an H-subunit deletion mutant) and SKΔLM (an (L+M) deletion mutant) of the purple non-sulphur bacterium Rhodospirillum rubrum have been investigated by femtosecond transient absorption spectroscopy. Upon excitation of the spirilloxanthin (Spx) S₂ state at 546 nm, of the bacteriochlorophyll Soret band at 388 nm and probing spectral regions, which are characteristic for carotenoids, similar dynamics in the SPUHK1, SKΔLM and WT strains could be observed. The excitation of Spx S₂ is followed by the simultaneous population of the lower singlet excited states S₁ and S* which decay with lifetimes of 1.4 and 5 ps, respectively for the mutants, and 1.4 and 4 ps, respectively, for the wild-type. The excitation of the BChl Soret band is followed by relaxation into BChl lower excited states which compete with excitation energy transfer BChl-to-Spx. The deexcitation pathway BChl(Soret) → Spx(S₂) → Spx(S₁) occurs with the same transition rate for all investigated samples (WT, SPUHK1 and SKΔLM). The kinetic traces measured for the Spx S₁ → S_N transition display similar behaviour for all samples showing a positive signal which increases within the first 400 fs (i.e. the time needed for the excitation energy to reach the Spx S₁ excited state) and decays with a lifetime of about 1.5 ps. This suggests that the Spx excited state dynamics in the investigated complexes do not differ significantly. Moreover, a longer excited state lifetime of BChl for SPUHK1 in comparison to WT was observed, consistent with a photochemical quenching channel present in the presence of RC. For long delay times, photobleaching of the RC special pair and an electrochromic blue shift of the monomeric BChl a can be observed only for the WT but not for the mutants. The close similarity of the excited state decay processes of all strains indicates that the pigment geometry of the LH1 complex in native membranes is unaffected by the presence of an RC and allows us to draw a model representation of the WT, SKΔLM and SPUHK1 PSU complexes.     

Time-resolved circular dichroism and absorption studies of the photolysis reaction of (carbonmonoxy)myoglobin.

Time-resolved circular dichroism (TRCD) and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin (MbCO). Following the spectral changes associated with the initial loss of CO, a subtle change is observed in the visible absorption spectrum of the Mb product on a time scale of a few hundred nanoseconds. No changes are seen in the CD spectrum of Mb in the visible and near-UV regions subsequent to the loss of CO. The data suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.