Effect of disordered hemes on energy transfer rates between tryptophans and heme in myoglobin.

Our recent linear dichroism study of heme transitions (Gryczynski, Z., E. Bucci, and J. Kusba. 1993. Photochem. Photobiology. in press) indicate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment in the near-UV region implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e., on the kappa 2 orientation parameter of the Förster equation. Using the atomic coordinates of SW myoglobin we have estimated the variation of kappa 2 parameter as a function of the heme absorption transition moment direction. The simulations proved that transfer is very efficient and anticipates lifetimes in the picosecond range. Also, they showed that transfer is very sensitive to rotations of the heme around its alpha-gamma-meso-axis, which may reduce the efficiency of transfer to almost zero values, producing lifetimes very similar to those of free tryptophan, in the nanosecond range. Comparisons between the lifetime values reported in the literature and those here estimated suggest that natural heme disorder, in which heme is rotated 180 degrees around its meso axis, is at the origin of the nanosecond lifetimes found in myoglobin systems.     

Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.

Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear.     

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.     

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.     

The conserved Trp-Cys hydrogen bond dampens the "push effect" of the heme cysteinate proximal ligand during the first catalytic cycle of nitric oxide synthase

Residues surrounding and interacting with the heme proximal ligand are important for efficient catalysis by heme proteins. The nitric oxide synthases (NOSs) are thiolate-coordinated enzymes that catalyze the hydroxylation of l-Arg in the first of the two catalytic cycles needed to synthesize nitric oxide. In NOSs, the indole NH group of a conserved tryptophan [W56 of the bacterial NOS-like protein from Staphylococcus aureus (saNOS)] forms a hydrogen bond with the heme proximal cysteinate ligand. The purpose of this study was to determine the impact of increasing (W56F and W56Y variants) or decreasing (W56H variant) the electron density of the proximal cysteinate ligand on molecular oxygen (O(2)) activation using saNOS as a model. We show that the removal of the indole NH···S(-) bond for W56F and W56Y caused an increase in the electron density of the cysteinate. This was probed by the decrease of the midpoint reduction potential (E(1/2)) along with weakened σ-bonding and strengthened π-backbonding with distal ligands (CO and O(2)). On the other hand, the W56H variant showed stronger Fe-OO and Fe-CO bonds (strengthened σ-bonding) along with an elevated E(1/2), which is consistent with the formation of a strong NH···S(-) hydrogen bond from H56. We also show here that changing the electron density of the proximal thiolate controls its "push effect"; whereas the rates of both O(2) activation and autoxidation of the Fe(II)O(2) complex increase with the stronger push effect created by removing the indole NH···S(-) hydrogen bond (W56F and W56Y variants), the W56H variant showed an increased stability of the complex against autoxidation and a slower rate of O(2) activation. These results are discussed with regard to the roles played by the conserved tryptophan-cysteinate interaction in the first catalytic cycle of NOS.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

Hydrophobic clustering in acid-denatured IL-2 and fluorescence of a Trp NH-pi H-bond

The single tryptophan at position 121 of human interleukin-2 (IL-2) can form an NH-pi hydrogen bond with Phe 117 involving the indole nitrogen and the benzene aromatic ring. At pH 5.5, this type of aromatic interaction results in a fluorescence quantum yield three-fold lower than that of a fully solvent exposed tryptophan. At pH 2.1, IL-2 forms a compact denatured state with twice the emission intensity of the native protein. Global analysis of time-resolved fluorescence emission at multiple emission wavelengths shows that native and acid-denatured IL-2 can be described by four decay components. The fractional amplitudes of the shortest sub-nanosecond lifetimes are higher in the native state, suggesting rapid quenching due to the NH-pi hydrogen bond. In the denatured state, longer lifetimes have greater fractional amplitudes, indicating a smaller population of hydrogen-bonded species. Electrostatic-dipolar relaxation of the tryptophan microenvironment upon excitation is greater in the native-state of IL-2 than the acid-denatured state. This suggests that acid-denaturation sequesters Trp 121 from polar residues, while maintaining an interaction with Phe 117. This is consistent with the model of secondary structure preservation and hydrophobic clustering in molten-globule intermediates.     

Tryptophan side chain conformers monitored by NMR and time-resolved fluorescence spectroscopies

We have inserted a tryptophan (F77W) in the core of the regulatory domain of cardiac troponin C (cNTnC), and previously determined the structure of this mutant with and without the cosolvent trifluoroethanol (TFE). Interestingly, the orientations of the indole side chain of the Trp are in opposite directions in the two structures (Julien et al., Protein Sci 2009; 18:1165-1174). Fluorescence decay experiments for single Trp-containing proteins often show several lifetimes, which have been interpreted as reflecting conformational heterogeneity of the Trp side chain resulting from different rotamers. To test this interpretation, we monitored the effect of TFE on wild type, F77W and F77W-V82A calcium-saturated cNTnC using 2D (13)C-HSQC NMR and time-correlated single photon counting fluorescence spectroscopies. The time dependence of the Trp fluorescence decay was fit with three lifetimes. Addition of TFE caused a gradual, but limited decrease of the lifetimes due to dynamic quenching. For F77W cNTnC, the amplitude fractions of the lifetimes also changed upon addition of TFE-the long lifetime increased from 13 to 29%, while the middle lifetime decreased from 63 to 50% and the short lifetime remained relatively unchanged. For F77W-V82A cNTnC, comparable NMR changes are observed, confirming the switch in rotamer conformation, but only much smaller changes in fluorescence decay parameters were detected. These data indicate that the balance between the rotamer states can be changed without changing the lifetime amplitude fractions appreciably, and suggest that the environment(s) of the indole ring, responsible for the different lifetimes, can result from factors other than the intrinsic rotamer state of the tryptophan.     

Polarized absorption picosecond kinetics as a probe of energy transfer in phycobilisomes of Synechococcus 6301

The energy transfer between C-phycocyanin chromophores in intact phycobilisomes of Synechococcus 6301 is shown to lead to an anisotropy relaxation with a lifetime of 10 ± 2 ps. However, due to the molecular order within the hexameric units of C-phycocyanin the anisotropy does not decay to zero. The Förster dipole-dipole mechanism of energy transfer can qualitatively explain these data provided that there is no back transfer of excitation energy and that the chromophore distribution is non-random. The rate of energy transfer in phycobilisomes between C-phycocyanin and allophycocyanin can best be described by a double exponential with lifetimes of 12 ± 3 and 84 ± 8 ps.     

Ligand binding to a hemoprotein lacking the distal histidine. The myoglobin from aplysia limacina (Val(E7))

The time course of ligand recombination to the myoglobin from Aplysia limacina, which has Val(E7), was measured following photolysis by flashes of 35 ps to 300 ns with a time resolution of 10 ps or 1 ns. CO shows only biomolecular recombination. O2 has a small geminate reaction with a half-time of tens of picoseconds, but no nanosecond geminate reaction. NO has two picosecond relaxations with half-times of 70 ps (15%) and 1 ns (80%) and one nanosecond relaxation with a half-time of 4.6 ns. The biomolecular rates for O2 and NO are the same: 2 x 10(7) M-1 s-1. Methyl and ethyl isonitriles have a geminate reaction with a half-time of 35 ps. Ethyl isonitrile has, in addition, a nanosecond relaxation (25%) with a half-time of 100 ns. t-Butyl isonitrile has four geminate relaxations (10 ps, 35 ps, 1 ns, and 1 microseconds). Analysis of the results suggests much easier movement of ligand between the heme pocket and the exterior than in sperm whale myoglobin (His(E7]. The reactivity of the heme is little different, placing the effect of the differences from sperm whale myoglobin on the distal side of the heme.     

Age-related changes in the biomechanics of left ventricular twist measured by speckle tracking echocardiography

The increasing number and proportion of aged individuals in the population warrants knowledge of normal physiological changes of left ventricular (LV) biomechanics with advancing age. LV twist describes the instantaneous circumferential motion of the apex with respect to the base of the heart and has an important role in LV ejection and filling. This study sought to investigate the biomechanics behind age-related changes in LV twist by determining a broad spectrum of LV rotation parameters in different age groups, using speckle tracking echocardiography (STE). The final study population consisted of 61 healthy volunteers (16-35 yr, n=25; 36-55 yr, n=23; 56-75 yr, n=13; 31 men). LV peak systolic rotation during the isovolumic contraction phase (Rot(early)), LV peak systolic rotation during ejection (Rot(max)), instantaneous LV peak systolic twist (Twist(max)), the time to Rot(early), Rot(max), and Twist(max), and rotational deformation delay (defined as the difference of time to basal Rot(max) and apical Rot(max)) were determined by STE using QLAB Advanced Quantification Software (version 6.0; Philips, Best, The Netherlands). With increasing age, apical Rot(max) (P<0.05), time to apical Rot(max) (P<0.01), and Twist(max) (P<0.01) increased, whereas basal Rot(early) (P<0.001), time to basal Rot(early) (P<0.01), and rotational deformation delay (P<0.05) decreased. Rotational deformation delay was significantly correlated to Twist(max) (R(2)=0.20, P<0.05). In conclusion, Twist(max) increased with aging, resulting from both increased apical Rot(max) and decreased rotational deformation delay between the apex and the base of the LV. This may explain the preservation of LV ejection fraction in the elderly.     

Interfacial tryptophan residues: a role for the cation-pi effect?

Integral membrane proteins are characterized by having a preference for aromatic residues, e.g., tryptophan (W), at the interface between the lipid bilayer core and the aqueous phase. The reason for this is not clear, but it seems that the preference is related to a complex interplay between steric and electrostatic forces. The flat rigid paddle-like structure of tryptophan, associated with a quadrupolar moment (aromaticity) arising from the pi-electron cloud of the indole, interacts primarily with moieties in the lipid headgroup region hardly penetrating into the bilayer core. We have studied the interaction between the nitrogen moiety of lipid molecule headgroups and the pi-electron distribution of gramicidin (gA) tryptophan residues (W9, W11, W13, and W15) using molecular dynamics (MD) simulations of gA embedded in two hydrated lipid bilayers composed of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoylphosphatidyl-choline (POPC), respectively. We use a force field model for tryptophan in which polarizability is only implicit, but we believe that classical molecular dynamics force fields are sufficient to capture the most prominent features of the cation-pi interaction. Our criteria for cation-pi interactions are based on distance and angular requirements, and the results from our model suggest that cation-pi interactions are relevant for W(PE)1), W(PE)13, W(PE)15, and, to some extent, W(PC)11 and W(PC)13. In our model, W9 does not seem to engage in cation-pi interactions with lipids, neither in POPE nor POPC. The criteria for the cation-pi effect are satisfied more often in POPE than in POPC, whereas the H-bonding ability between the indole donor and the carbonyl acceptor is similar in POPE and POPC. This suggests an increased affinity for lipids with ethanolamine headgroups to transmembrane proteins enriched in interfacial tryptophans.     

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

A cysteine was introduced into the FG-loop (P187C) of CYP51 from Mycobacterium tuberculosis (MT) for selective labeling with BODIPY and fluorescence energy transfer (FRET) analysis. F?rster radius for the BODIPY-heme pair was calculated assuming that the distance between the heme and Cys187 in solution corresponds to that in the crystal structure of ligand free MTCYP51. Interaction of MTCYP51 with azole inhibitors ketoconazole and fluconazole or the substrate analog estriol did not influence the fluorescence, but titration with the substrate lanosterol quenched BODIPY emission, the effect being proportional to the portion of substrate bound MTCYP51. The detected changes correspond to approximately 10A decrease in the calculated distance between BODIPY-Cys187 and the heme. The results confirm (1) functional importance of conformational motions in the MTCYP51 F/G segment and (2) applicability of FRET to monitor them in solution.     

Phosphorescence and optically detected magnetic resonance of 4',6-diamidino-2-phenylindole (DAPI) and its complexes with [d(CGACGTCG)]2 and [d(GGCCAATTGG)]2

Phosphorescence and optical detection of magnetic resonance (ODMR) is used to study the excited triplet state of 4',6-diamidino-2-phenyl indole (DAPI) and its complexes with the oligonucleotides [d(CGACGTCG)](2) and [d(GGCCAATTGG)](2), where binding occurs by intercalation between GC base pairs and by minor groove insertion, respectively. Weaker binding of DAPI to phosphate is also detected, and the triplet state of this complex is characterized. Intercalation with [d(CGACGTCG)](2) produces a phosphorescence redshift, while groove binding with [d(GGCCAATTGG)](2) leads to a blueshift. Both binding modes give rise to a small decrease in the zero-field splitting (zfs) of the DAPI triplet state. The largest redshift and zfs decrease are found for the phosphate complex. The phosphorescence lifetimes are shorter by an order of magnitude than that of indole or tryptophan as expected for the lower triplet state energy, E(00), of DAPI. The lifetimes agree well with a correlation with E(00) introduced by Siebrand [Siebrand, W. (1966) J. Chem. Phys. 44, 4055-4057] except for the [d(GGCCAATTGG)](2) minor groove complex with a lifetime that is about 20% too long. The longer lifetime is attributed to distortion of the amidino groups in this complex, resulting in less efficient intersystem crossing.     

Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (~20–70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (~15 Å), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model.     

Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit.

Quenching of the room-temperature phosphorescence of Escherichia coli alkaline phosphatase by several freely diffusing molecules was studied, each of whose absorption spectrum overlaps the long-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced F?rster energy transfer. The presence of additional nonresonance transfer mechanisms was also detected, from a lack of linear dependence of quenching rate on spectral overlap. The quenching agents used were the dye molecules methyl red, methyl orange, and 2-[(4-hydroxyphenyl)azo]benzoic acid, as well as the embedded heme groups of myoglobin, metmyoglobin, and the reduced and oxidized forms of cytochrome c. Quenching was found to be greatly diminished upon reduction of each acceptor, indicating that electron transfer occurs efficiently from the excited tryptophan to the oxidized form of the acceptors. The elimination of this electron transfer in the reduced form affords the opportunity to separately measure the F?rster transfer rates for the heme proteins. When the transfer rate constant thus measured for myoglobin is applied to a model where both donor and acceptor proteins are taken to be spherical with both tryptophan and the heme group placed off center (a model whose quenching rate equation is newly presented here), the depth of the phosphorescent tryptophan beneath the surface of alkaline phosphatase is found to be 16 A. This value is close to the depth of tryptophan 109 (which is known to be the phosphorescent residue in alkaline phosphatase), showing that with properly chosen probes this technique is indeed valuable for distance determinations in protein structure studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation factor in steady-state and time-resolved resonance energy transfer measurements.

Resonance energy transfer measurements provide a way to estimate distances between chromophores attached to different sites of macromolecules. There are two unknowns involved in resonance energy transfer measurements, the distance between two chromophores and their relative orientation. When static orientational disorder exists, the orientation factor, kappa 2, can vary from 0 to 4, leading to considerable uncertainty in estimation of distances. Fluorescence polarization anisotropy measurements can reduce the degree of uncertainty [Dale & Eisinger (1974) Biopolymers 13, 1573]. There may still be substantial error bounds for the average distance measurements. Time-resolved fluorescence measurements provide an "apparent" average distance and distance distribution containing contributions by both distance and orientation. The contribution of orientation to observed "apparent" average distance and distance distribution widths has been estimated for both simulated and real data. With a single unique distance as input in the simulation and with random but static orientation of donor and acceptor, the recovered average distance is very close to that of the input when the input distance is close to or larger than the F?rster distance. The recovered width of apparent distance distribution can be substantial and it changes as a function of F?rster distance to average distance ratio and as a function of F?rster distance. Similar conclusions apply to the case where there is a real distance distribution. Motional averaging of the orientation was simulated by the Monte Carlo method to estimate the contribution of orientation when chromophores have certain degrees of mobility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triplet-singlet energy transfer in the complex of auramine O with horse liver alcohol dehydrogenase

Triplet-singlet energy transfer has been studied in the complex formed between auramine O (AO) and horse liver alcohol dehydrogenase with optically detected magnetic resonance (ODMR) spectroscopy. The results show that Trp-15 and Tyr residues transfer triplet energy mainly by a trivial process, whereas Trp-314 transfers triplet energy by a F?rster process with two observed lifetimes at 77 K of 170 and 50 ms. The different F?rster energy-transfer lifetimes are ascribed either to quenching of the two Trp-314 residues of the dimer by a single asymmetrically bound AO or to two distinct conformations of the enzyme-dye complex with differing separations and/or orientations of donor and acceptor. Individual spin sublevel transfer rate constants are reported for the major decay component with the 170-ms Trp triplet-state lifetime; these are found to be highly selective with kxtr much greater than kytr and kztr.     

Steady-state and picosecond time-resolved fluorescence studies on native and apo seed coat soybean peroxidase.

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase super family. The protein has a very similar 3-dimensional structure with that of horseradish peroxidase (HRP-C). The fluorescence characteristics of the single tryptophan (Trp117) present in SBP and apo-SBP have been studied by steady-state and pico-second time-resolved fluorescence spectroscopy. Fluorescence decay curve of SBP was best described by a four exponential model that gave the lifetimes, 0.035 ns (97.0%), 0.30 ns (2.0%), 2.0 ns (0.8%), and 6.3 ns (0.2%). These lifetime values agreed very well with the values obtained by the model independent maximum entropy method (MEM). The three longer lifetimes that constituted 3% of the fluorophore population in the SBP sample are attributed to the presence of trace quantities of apo-SBP. The pico-second lifetime value of SBP is indicative of efficient energy transfer from Trp117 to heme. From fluorescence resonance energy transfer (FRET) calculations, the energy-transfer efficiency in SBP is found to be relatively higher as compared to HRP-C and is attributed mainly to the higher value of orientation factor, kappa(2) for SBP. Decay-associated spectra of SBP indicated that the tryptophan of SBP is relatively more solvent exposed as compared to HRP-C and is attributed to the various structural features of SBP. Linear Stern-Volmer plots obtained from the quenching measurements using acrylamide gave k(q) = 5.4 x 10(8) M(-1) s(-1) for SBP and 7.2 x 10(8) M(-1) s(-1) for apo-SBP and indicated that on removal of heme in SBP, Trp117 is more solvent exposed.     

Sub-nanosecond photolysis studies of Fe protoporphyrin IX solubilized in neat dimethyl sulfoxide

The present study examines the optical properties of the sub-nanosecond photolytic transient of Fe(II)protoporphyrin IX (Fe(II)PPIX) in neat dimethyl sulfoxide (DMSO). Previous nanosecond studies have revealed that photolysis of the (DMSO)₂Fe(II)PPIX complex in neat DMSO results in the formation of a five-coordinate high-spin (DMSO)Fe(II)PPIX complex within ∼100 ns which decayed with a pseudo-first order rate constant of 2 × 10⁶ s⁻¹ (Larsen et al. (1995) [19]). The results presented here demonstrate that the five-coordinate (DMSO)Fe(II)PPIX species is generated in <100 ps and that no significant changes occur in the kinetic difference between 100 ps and ∼100 ns. The 100 ps transient spectrum of the (DMSO)Fe(II)PPIX complex was also constructed from the kinetic difference spectrum and the equilibrium spectrum of the (DMSO)₂Fe(II)PPIX. The 100 ps transient spectrum exhibits a Soret maximum at ∼432 nm close to that of deoxyMb (435 nm, imidazole coordination) consistent with S-bonded DMSO occupying the fifth coordination site. Neither base elimination is detected on time scales down to 100 ps nor is there evidence for transient O-bonded DMSO followed by linkage isomerization to the equilibrium S-bonded form. The unusually slow rate of DMSO recombination is attributed to electrostatic interactions between DMSO and the five-coordinate heme iron as well as intermolecular interactions between solvent molecules in the bulk, as has been previously proposed.