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The photoexcited metastable triplet state of Mg2+-mesoporphyrin IX (MgMPIX) or Mg2+-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg2+ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8–110K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MgPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is ≫150cm−1, suggesting symmetry breaking from the anisotropy of internal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527–16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q0,0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg2+ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.

The absorption and emission properties of hybrid Zn-substituted human hemoglobin (Hb) were used to monitor differences in interaction between the porphyrin and the polypeptide chain for the two subunits. Although alpha-substituted (alpha-ZnHb), beta-substituted (beta-ZnHb), or totally substituted Hb all show optical properties characteristic of Zn porphyrins, the spectra are also indicative of specific interactions between the polypeptide chain and the porphyrin. The Q0,0 absorption band of alpha-ZnHb at 5 K shows a splitting of approximately 300 cm-1, comparable to the largest split ever reported for a heme protein. This value is approximately 140 cm-1 for beta-ZnHb. The possible origin of the split is discussed in terms of the local electric field imposed by the amino acids of the respective heme pockets, different configurations of the porphyrin, and/or influences of the liganding histidine. The Zn derivatives show quasiline spectra under fluorescence line narrowing conditions, and the resolved excitation spectrum reveals differences in the vibrational levels of the Zn porphyrin in the two subunits. Broad underlying emission in the fluorescence line-narrowed emission spectrum can be accounted for, in part, by the existence of the two closely spaced electronic origins and also by the extent of phonon coupling between the porphyrin and the protein matrix.

The FTIR spectra of the asymmetric carboxylate absorption region of three c -type cytochromes—namely horse heart, yeast and bonito cytochromes c —as well as continuum electrostatic calculations performed on their respective protein matrices, show that these combined methods can target specific protein regions and yield pertinent protein charge information that correlates with the observed spectral data. Deconvolution of the IR carboxylate stretch frequency region (1525–1675 cm −1 ) in the three cytochromes yield different ν oco a distributions. In the case of the bonito cytochrome c carboxylates, two ν oco a populations are clearly distinguishable in the deconvoluted spectra—which is not the case for the more complex ν oco a deconvolutions of the other two cytochromes. The frequency distributions of the calculated potentials are consistent with the experimental observations and we conclude that the IR carboxylate absorption in proteins can be modified by the electrostatic environment.

The influence of the protein on the nonplanarity of the macrocycle for nickel(II)-reconstituted cytochrome c (NiCyt-c) has been investigated with pH-dependent resonance Raman and UV-visible absorption spectroscopy and molecular mechanics calculations. The spectra reveal that NiCyt-c near neutral pH has axially coordinated Ni, but below pH 3 and above pH 12, four-coordinate species predominate. The shape of the structure-sensitive Raman line nu10 of NiCyt-c is asymmetric and broad and it changes with pH. This broad line can be decomposed well into at least two sublines, a low-frequency line that results from a nonplanar conformer and a high-frequency line that arises from a nearly planar conformer. Upon lowering the pH from 3.0 to 1.0, the amount of the nonplanar conformer decreases relative to that of the planar conformer. The decreased nonplanarity can be accounted for in terms of the disruption of a hydrogen-bonding network in the peptide backbone upon lowering the pH. Molecular mechanics (MM) calculations on iron(III) and nickel(II) microperoxidase 5 (MP-5) as well as some model heme derivatives have been carried out in order to locate the part of the protein that causes the heme distortion observed in the X-ray crystal structures of cytochromes c. The energy-optimized structures of MP-5 and the model compounds were analyzed using the normal-coordinate structural decomposition method to specify and quantify the out-of-plane macrocyclic distortions. MM calculations for MP-5 show that two hydrogen bonds formed between the amide groups in the peptide backbone are important in maintaining the ruffled deformation of the macrocycle. All evidence presented supports the hypothesis that the nonplanar distortion of the porphyrin of cytochromes c is largely maintained by a relatively small protein segment including the cysteines, the amino acids between the cysteines, and the adjacent histidine ligand. Hydrogen bonding within the backbone of this segment is important in maintaining the conformation of the peptide that induces the porphyrin distortion.

The carboxylate groups of organic acids give strong absorption in the infrared between approximately 1550 and 1650 cm-1. For acetate and chloroacetate derivatives, the infrared (IR) frequency of the carboxylate antisymmetric stretching mode (v(a)OCO) is related to the square root of the pK of the acid, with a shift of approximately 20 cm-1 to higher frequency for a pK drop in the range 5-3. It follows that v(a)OCO may respond to conditions on the protein surface. In this paper, the IR amide I' and carboxylate absorptions of cytochrome c from horse, yeast, and tuna are compared with model compounds such as Val-Glu and microperoxidase-11, the 11 amino acid fragment of horse cytochrome c containing the covalently bound heme. For microperoxidase-11, the contribution from all four carboxylates can be accounted for and the 1567 cm-1 absorption is assigned to the heme propionates. For the proteins, the carboxylate absorption band is inhomogeneous, i.e., there is a distribution of frequencies. Both the amide I' and carboxylate bands are sensitive to protein conformation as shown by their different pH, salt, and redox dependence.

Perhaps the most important contribution of FLN is that it provides an experimental approach to relate physical changes in the protein to predicted dynamical behavior. It is clear that the sample is inhomogeneously broadened in a continuous manner, consistent with the damped motion of proteins. At the same time configurational substates can be selected, suggesting that there is indeed a hierarchy of protein motion and structure. As yet, identification of the structure, and relating it to the spectra, has not been achieved. It is clear that the electric field exerted by neighboring atoms shifts the electronic transition, and the inhomogeneity is greater when the surrounding disorder is greater. The inhomogeneity for the chromophore in the protein is dependent on the protein conformation and is intermediate between that of a crystal and a glass. The phonon coupling also depends on the chromophore and the protein. Fluorescence line narrowing provides in addition ground- and excited-state vibrational frequencies, thereby allowing for structural differences between the excited-state and the ground-state molecule to be detected.

The alpha or Q0,0 absorption band of horse iron(II) cytochrome c splits and shifts to the blue as temperature decreases over the temperature range of 290-10 K. At room temperature, its maximum is at 18 150 cm-1 and the spectral width is 273 cm-1, whereas at 10 K, the two bands of the Q0,0 transition occur at 18 364 and 18 253 cm-1 and the width of the lowest-energy band is 96 cm-1. Temperature dependent splitting also occurs for zinc cytochrome c, a derivative in which Fe has been replaced by Zn; at 10 K, the peaks in the Q0,0 band region occur at 17 106 and 16 996 cm-1. The peak positions are independent of the cryosolvent (aqueous ethylene glycol or glycerol mixtures). The splitting of the Q0,0 band seen in the protein (approximately 110 cm-1 for iron and zinc cytochrome c) is comparable to the crystal field splitting observed for metalloporphyrins in mixed crystals. In contrast, the Q0,0 band of zinc coproporphyrin III in a glassy solvent (dimethylformamide/ethylene glycol) or in poly(vinyl chloride) shows a blue shift with temperature decrease but no evidence of Q0,0 splitting. Available spectral data show that the Q0,0 band is composed of two nearly degenerate electronic transitions and the split is due to the asymmetry in the heme pocket of the protein that arises from the surrounding polypeptide chain. This asymmetry results in the stabilization of one form of the excited state over the other, according to a Jahn-Teller mechanism.

The cyanide complex of FeIIMb prepared and maintained at temperatures below 0 degrees C is sufficiently stable to permit spectroscopic characterization and allow comparison with free HCN and other ferric and ferrous CN complexes. The visible absorption spectrum of FeIIMb-CN has a split alpha band maxima at 571 and 563 nm, suggesting distortion in the x-y plane of the porphyrin. FeIIMb-CN, like the CO complex, was found to be optically active by circular dichroism. The C-N stretching frequencies for the CN-ferrous complexes are very sensitive to parameters within the heme pocket. The values are as follows: FEIIMb at pH 8, 2057 cm-1 with a shoulder appearing at 2078 cm-1 at pH 5.6; FeIIMp, 2034 cm-1. In contrast, the frequencies for C-N stretch differ little among ferric heme complexes, ranging from 2123 to 2125 cm-1 for myoglobin, hemoglobin, and microperoxidase. These values compare with free HCN (2094 cm-1) or CN- (2080 cm-1). Quantum chemical modeling of the neutral iron-porphyrin complex with imidazole and cyanide and of its anion was used to explain the effects of the cyanide coordination and of iron reduction on the C-N stretching frequencies. The lower nu C-N for FeIIMb-CN relative to the ferric complex is attributed to the appearance of additional electron density on all the anti-bonding CN orbitals. The extra electron density was also used to explain that the band width of C-N stretching mode was greater in the ferrous complexes than in the ferric complex. Finally, the calculation shows that sigma donation weakens the Fe-C bond, in qualitative agreement with the spontaneous dissociation of CN- from FeIIMb at -5 degrees C. The sensitivity of CN complexes of ferrous heme proteins to the heme pocket environment and the ability to correlate spectroscopic parameters with calculated electron density suggest that infrared spectroscopy of the CN ligand is an appropriate tool to study ferrous heme proteins.

The involvement of near-UV light in cataract development suggests that potential anti-cataract drugs may display unusual spectroscopic properties. As bendazac impedes certain effects associated with lens opacification, we have characterized the singlet and triplet states of bendazac and its analog, benzydamine, by fluorescence and phosphorescence methods. These compounds have much shorter triplet state lifetimes compared to the triplet state lifetimes observed in proteins. Our results raise the possibility that the photoprotective action of these compounds may result from their ability to dissipate energy through the triplet state. We propose alternative modes for the photoprotective actions of these compounds.

Fluorescence emission and triplet-triplet absorbance spectroscopy of the single tryptophan in cod parvalbumin were used to study the stability and dynamics of the protein as influenced by Ca2+ binding and interaction with a chaotropic agent. The concentrations for half-saturation for Ca binding were 3.6 x 10(-9), 3.3 x 10(-4), 7.1 x 10(-3), and 0.14 M in the presence of 0, 2, 3, and 4 M guanidine hydrochloride, respectively. As predicted for thermodynamic reversibility, the guanidine hydrochloride unfolding reaction depends upon Ca2+, and the delta G are as follows: 22.9, 29.3, 35.2, and 44.2 kJ/mol for no added Ca2+, 1, 2, and 5 mM Ca2+, respectively. The stability toward denaturation imparted by the binding of two Ca2+ is about -60 kJ/mol. For Ca(2+)-bound parvalbumin in the presence of excess Ca2+, the decay of the triplet state tryptophan is approximately exponential, and the lifetime decreases from 6.5 to 3.8 ms as the temperature increases from 10 to 40 degrees C. In contrast, the triplet decay of the calcium-free protein is nonexponential over the time range of microseconds to milliseconds, a result that may indicate that the Ca-free protein is molten-globule-like. At Ca2+ concentrations where the protein is partially saturated with Ca2+, the lifetime of the longest decay component is less than that for the Ca-saturated protein; this finding suggests an exchange of Ca2+ and a conformational change during the triplet lifetime. From these data, a rate constant for the process that includes calcium-related protein conformational change can be surmised to range between 200 and 500 s-1.

Mesoporphyrin IX was used as a fluorescent analogue of heme in cytochrome c peroxidase (CcP). Details of the fluorescence spectra of CcP obtained under conditions of energy selection revealed interactions of the porphyrin with the heme pocket. It was shown that the energy of a 0,0 transition shifted with pH in parallel with changes in the electric field of the protein.

Electronic spectra of mesoporphyrin-substituted yeast cytochrome c peroxidase (MP-CcP) were measured as a function of pH, ionic strength, and binding of cytochrome c (cyt c) by fluorescence line narrowing (FLN) spectroscopy at 5 K. The FLN spectra provided information about the vibrational structure of the first excited singlet state of MP-CcP, the various tautomeric forms of mesoporphyrin, and the positions and widths of their 0,0 bands. The composite 0,0 band of MP-CcP at pH 6 could be resolved into three components with peak positions at 16,046, 16,103, and 16,203 cm-1. MP-CcP at pH 8 could be analyzed using two components with peak positions at 16,048 and 16,193 cm-1. The disappearance of the 16,103-cm-1 component at alkaline pH suggests that it is due to a "chemical substate" arising from protonation of His52 in the distal side of the porphyrin. Computer simulations of the electrostatic field that CcP imposes on its porphyrin show that, in the presence of charged axial histidines His52 and His175, the electrostatic field at porphyrin nitrogens increases, especially along the normal to the heme by about 200 mV/A. Electric field effects may account for pH-dependent spectral shifts of the 0,0 positions of the resolved components, although hydrogen bonding may also affect these positions. On the other hand, the peak position of the components was not affected by ionic strength or binding of cyt c, implying that the electrostatic field of the heme pocket of MP-CcP remains unchanged. Indeed, computed changes in ionic strength of the solvent show no modification of the electrostatic field at the porphyrin. The only detectable effect of ionic strength and binding of cyt c to MP-CcP is on the relative contributions of the components, suggesting some rearrangements in the vicinity of the heme. Finally, shifts in the position of the vibrational lines for MP-CcP components indicate either that the tautomers have different vibrational frequencies due to the nonsymmetry of the porphyrin and/or that tautomers experience various distortions. Comparison of the vibrational spectrum of the first excited singlet state of mesoporphyrin in CcP and horseradish peroxidase also suggests that the heme pocket in the two peroxidases provides different steric restrictions.

We investigated spectral holes burnt at 1.5 K into the origins of several tautomeric forms of mesoporphyrin IX-substituted horseradish peroxidase at pH 8 under pressures up to 2 MPa. From the pressure-induced lineshift the compressibility of the apoprotein could be determined. We found that the compressibility changed significantly when measured at different tautomer origins. It was concluded that there must be a correlation between the tautomer configurations of the chromophore and the actual structures of the apoprotein. As a consequence, specific conformational substates of the protein can be selected by optical selection of the associated tautomers.

The porphyrin in metal-free cytochrome c molecules was investigated using fluorescence line narrowing spectroscopy. An energy map is defined to illustrate the environmental effects on the vibronic structure of a chromophore. In the approximation of pure electronic distortion, the population distribution in the energy scale can be characterized by a function. An intuitive definition of this distribution function is determined for the porphyrin in cytochrome c, and the validity of this approximation is experimentally verified. In addition, numerous excited state vibrational levels suggest a method to quantify the relative absorption probabilities for (0,0)yields(1,k) transitions.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Oxygen is the primary oxidant in energy-producing biological reactions and is also involved in the synthesis and degradation of many structural and regulatory molecules of physiological importance. This review discusses the advantages and limitations of the currently available methods for measuring oxygen in mammalian tissue and bodily fluids. These include 1) the effects of O2 on the relaxation time of molecules excited by electromagnetic radiation and observed by optical (fluorescence and phosphorescence) and magnetic (nuclear magnetic resonance and electron paramagnetic resonance) techniques, 2) the polarographic and galvanic reduction of oxygen at metal surfaces, 3) in vivo spectrophotometry of intrinsic redox systems, 4) manometry and tonometry, and 5) mass spectroscopy. The values of tissue oxygenation obtained with these techniques are compared with the Michaelis constant values for oxygen of almost 60 oxygen-consuming enzymes involved in mammalian tissue metabolism.

Fluorescence line narrowing (FLN) spectroscopy was used to study the role of the polypeptide chain in influencing the spectrum of Zn-substituted cytochrome c (Zn cyt c) and metal-free cyt c (porphyrin cyt c). For both derivatives the spectra show characteristics of relaxed fluorescence from an inhomogeneously broadened sample. Zero phonon lines and phonon wings can be clearly distinguished, and vibrational frequencies of the ground and excited states were identified. The inhomogeneous distribution width for porphyrin cyt c is slightly wider than that of Zn cyt c and a second population of molecules was apparent in the porphyrin cyt c. The phonon coupling was greater for Zn cyt c than for porphyrin cyt c, which may be due to the extra coupling to the polypeptide chain by metal ligation.

Anaerobic solutions of lens alpha-crystallin were subjected to near-UV (greater than 295 nm) irradiation, and the photoproducts were analyzed by fluorescence and room-temperature phosphorescence spectroscopy. The principal photoproduct was excited maximally at 340 nm, fluoresced maximally at 430 nm, and phosphoresced with an emission maximum at 510 nm. The phosphorescence intensity decay of this species was well fit by a sum of two exponentials with lifetimes of 9.2 ms (78%) and 61 ms (22%); this report is the first demonstration of a long-lived triplet state associated with a protein photolysis product. As reported previously, 3trp* is also long-lived in deoxygenated alpha-crystallin solution at room-temperature (Berger and Vanderkooi, 1989, Biochemistry 28, 5501-5508), hence both tryptophan and photoproduct triplet states are good candidates to mediate photodamage. Photolysis experiments in the presence of agents known to alter the tryptophan triplet yield provide evidence for the importance of triplet-state-mediated photodamage of lens crystallins in anaerobic solution. In 30 mM acrylamide where 3trp*, but not 1trp*, is efficiently quenched, anaerobic solutions exhibited marked resistance to protein photodamage, whereas the photoprotection in aerobic solution was minimal. In D2O, where photoionization is suppressed but triplet states are longer-lived, photodamage was accelerated in anaerobic solution but reduced in aerobic solutions. Finally, the anaerobic photodestruction rate was increased in 500 mM Cs+ solution where the triplet yield is increased by a heavy atom effect.

The small-molecule (i.e. oxygen) penetrability into proteins carries important implications for both the structural character and dynamic properties of proteins. We investigated aspects of protein dynamics by studying triplet-state quenching of Zn and Pd derivatives of myoglobin by oxygen. These two derivatives make an interesting comparison because the Zn derivative of myoglobin is five coordinated and is out-of-plane (as is deoxy myoglobin), whereas Pd porphyrin is planar (as is oxy myoglobin). Therefore relaxation of the polypeptide chain in the excited state analogues of the two functional conformations of myoglobin can be studied. In case of Znmyoglobin, the spectra did not exhibit an isosbestic point and the decay kinetics to the ground state dependtd upon the wavelength of measurement. The decay profiles could be fit by a double exponential function with a good correlation. In the case of Pd-myoglobin an isosbestic point was observed in the emission and the decay profile could be fit with a single exponential function. The comparison of the quenching constants for the two metal substituted derivatives suggests that there is no significant difference between the oxygen penetrability of the Zn and the Pd substituted myoglobins.

A novel technique for modelling intramolecular energy transfer is presented. Brownian dynamics calculations are used to compute the trajectories of donor and acceptor species, and the instantaneous orientation factor is calculated during each temporal iteration. In this work, several model systems are considered. Trajectories were computed for energy transfer between a flexible donor and a rigidly fixed acceptor. We have considered configurations where the donor is, (1) tethered to a fixed point in space, but free to diffuse rotationally, and (2) constrained to wobble in a cone. The luminescence decay of the donor is ‘measured’, and a non-single-exponential decay is observed for configurations of efficient energy transfer. Luminescence anisotropy measurements of constrained and unconstrained donors reflect the contribution of both energy transfer and rotational diffusion to the shape of the anisotropy decay curve.

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.

The fluorescence emission of iron-free cytochrome c (0Cyt c) in a glassy matrix was investigated under conditions of very low temperature (4.2 K.) and narrow bandwidth laser excitation. Excitation into the vibronic band, Qx(1,0) resulted in highly resolved emission spectra of linewidth 10-20 cm−1. Using the model of selective excitation developed by Abram el al. (1975) and McColgan et al. (1978), the emission spectra of vibronic excitation afforded a method to investigate excited state vibrational structure. Furthermore, emission profiles have shown that in 0Cyt c, the site distribution (inhomogeneous broadening) has a width in the order of 200cm−1.

Fluorescent derivatives of cytochrome c were prepared by replacing the heme iron with closed-shell metals such as zinc or tin. The iron-free derivatives of cytochrome c bind to yeast lactate dehydrogenase (cytochrome b2) stoichiometrically and with high affinity. Spectral overlap exists between the fluorescence of porphyrin, Zn(II) or Sn(IV) cytochrome c and the absorption of the heme of cytochrome b; therefore dipole-dipole interaction is possible as predicted by Förster's theory of energy transfer. Changes in the fluorescence yield and the fluorescent decay profile of the cytochrome c derivatives are consistent with the view that the heme distance is sufficiently close for dipolar interactions. The distance calculated from the data depends upon assumptions in the theory for energy transfer and uncertainties in the experiment. It can be argued that due to the symmetry of the metalloporphyrins the relative orientations of the two hemes do not introduce a significant uncertainty in the calculation. However the decay profiles of the iron-free cytochromes are complex, possibly reflecting structural rearrangement of the polypeptide chain during the fluorescent lifetime. The steady-state fluorescent yields would indicate that the mean distance is around 1.8 nm.

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca 2+ , Mg 2+ , and Mn 2+ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current. On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca 2+ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca 2+ , Mg 2+ , or Mn 2+ are detected. A23187 produces fluorescent transients under conditions of Ca 2+ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca 2+ content. Changes in A23187 fluorescence due to mitochondrial Ca 2+ accumulation are much smaller by comparison and fluorescence transients are not detected. Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca 2+ and Mg 2+ , and less than 2 nsec in the presence of excess EDTA. The present results are consistent with a model of ionophore-mediated cation transport in which free M 2+ binds with A23187 at the membrane surface to form the complex M(A23187) + . Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187) 2 , which carries the divalent cation through the membrane. These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.

The midpoint redox potential of cytochrome c and the electron paramagnetic resonance spectra of nitroxide labeled cytochromes c were measured as a function of binding to purified cytochrome c oxidase, cytochrome c peroxidase, cytochrome b 5 and succinate—cytochrome c reductase. The midpoint redox potential of horse heart cytochrome c is lowered in the presence of cytochrome c oxidase and succinate-cytochrome c reductase, but is unchanged in the presence of cytochrome c peroxidase or cytochrome b 5 . Further evidence of binding is afforded by an increase in correlation time, T c , of the spin-labeled cytochrome c at methionine 65 upon binding to cytochrome c peroxidase, cytochrome c oxidase and succinate—cytochrome c reductase. The changes in midpoint redox potential and electron paramagnetic resonance spectrum of the spin-labeled derivative upon binding can either be the consequence of specific interaction leading to formation of ES complexes, or it can be due to nonspecific electrostatic interaction between positively charged groups on cytochrome c and negatively charged groups on the isolated cytochrome preparations.

The fluorescence parameters of ethenoadenosine derivatives are influenced by metal cations and pH, as summarized here. The pH profile of ethenoadenosine determined by fluorescence intensity gives a normal titration curve and is not affected by ionic strength. In contrast, the pH titration curves of etheno-ATP, etheno ADP, and etheno AMP depend upon ionic strength. At high ionic strength normal curves are obtained, whereas at low ionic strength anomalies are obtained; this suggests that the phosphates can interact with the ring, possibly by hydrogen binding to the ring nitrogens. The room temperature fluorescence of ethenoadenosine occurs from the base form, although excitation of either the acid or base forms can contribute to the emission. This result can be explained if the excited state pK is lower than the ground state pK, and if deprotonation occurs within the time scale of the excited state. At low pH values the fluorescence lifetime of the base form is dependent upon the buffer concentration, indicating that the reverse reaction, protonation, occurs. The affinity constants for the binding of metals to the ethenoadenosine phosphates resemble those for the corresponding adenosine phosphates. Ni(II) and Co(II) are more effective than Mn(II) in quenching the fluorescence of ethenoadenosine phosphates; this result is predicted by Förster's theory for energy transfer based upon the overlap between donor emission spectrum and acceptor absorption spectrum. The diamagnetic ions Mg(II), Ca(II), and Zn(II) do not appear to affect the fluorescence of the ethenoadenosine phosphates directly, but rather to affect the conformation of the molecule, thereby affecting the quantum yield.

Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6-phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.

Molecular motion of 1,6-diphenyl-1,3,5-hexatriene embedded in intact guinea pig alveolar macrophage membranes was investigated by using techniques of nanosecond timeresolved fluorescence anisotropy measurements in the temperature range of 0–50 °C, and as a function of benzyl alcohol concentration. It was shown that molecular arrangement and microheterogeneity of the hydrocarbon region surrounding 1,6-diphenyl-1,3,5-hexatriene molecules are dependent on the temperature and benzyl alcohol concentration. The lipid orientation order parameter, Sv, showed a discontinuity in the temperature range 12–40 °C, which may indicate a phase transition. N-Formylmethionylphenylalanine-stimulated production of O2− from macrophages increased with temperature parallel with changes in Sv. Benzyl alcohol decreases the magnitude of the lipid order parameter at all temperatures studied. In the same concentration range of benzyl alcohol, stimulated O2− production by macrophages was inhibited. These data show the complex relationship between lipid integrity in macrophage membranes and a physiological function of these cells. In addition, the results indicate that benzyl alcohol influences the integrity of both the protein and lipid hydrophobic regions of the membrane.

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.

The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesoporphyrin derivatives of horseradish peroxidases A and C, leghemoglobin, and myoglobin were examined as a function of temperature and excitation wavelength. At room temperature, the emission spectra were unresolved and were independent of excitation wavelength. At low temperature (4.2 K), the spectra depended upon excitation wavelength: using narrow-band excitation into the high-energy side of the 0-1 and 0-0 bands gave unresolved emission spectra whereas excitation into the low-energy side produced quasi-line spectra. The resolved spectra were different for the five proteins and further varied with pH, indicating chromophore-protein interactions. The spectra are interpreted in terms of site selection and phonon interactions.

Oxygen-dependent quenching of phosphorescence has been used to measure the dependence of mitochondrial oxidative phosphorylation on oxygen concentration in suspensions of isolated rat liver mitochondria. An instrument has been designed which simultaneously monitors the phosphorescence lifetime of a fluorophor and the reduction of cytochrome c by dual wavelength spectrophotometry. The phosphorescence lifetime method gives very rapid (less than 100 ms) measure of the oxygen concentration (Vanderkooi, J. M., Maniara, G., Green, T. J., and Wilson, D. F. (1987) J. Biol. Chem. 262, 5476-5482) from concentrations characteristic of air-saturated media to as low as 2 x 10(-8) M. The results may be summarized as follows. For well coupled rat liver mitochondria at pH 7.0 and in the presence of ATP, as the oxygen concentration was lowered, increased cytochrome c reduction was observed to begin at oxygen concentrations greater than 20 microM. For mitochondria in the presence of uncoupler, cytochrome c reduction began at oxygen concentrations less than 1.0 microM. The oxygen dependence of reduction of cytochrome c in well coupled mitochondria treated with ATP was strongly dependent on the pH of the suspending medium. Reduction of cytochrome c began at higher oxygen concentrations as the pH was made more alkaline. The oxygen concentration for half-maximal respiratory rates was much larger for well coupled mitochondria treated with ATP (approximately 0.7 microM) than for mitochondria treated with uncoupler (less than 0.1 microM). It is concluded that the oxygen dependence of mitochondrial oxidative phosphorylation is such that mitochondria could function in their proposed role of tissue oxygen sensors for regulation of such diverse functions as local blood flow and electrical activity in the carotid body.

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.

Fluorescence site selection spectra of Zn‐substituted cytochrome c and Zn‐mesoporphyrin were measured as a function of excitation energy and temperature within 5–60 K. The emission energies determined for Zn‐cytochrome c were comparable with those of Zn‐mesoporphyrin and mesoporphyrin in polar glassy matrices and with resonance Raman spectra of heme proteins. In the site selection spectra of both Zn‐cytochrome c and Zn‐mesoporphyrin in polar organic glass an increase in the intensity of zero phonon emission lines was observed with an increase of temperature between 5 and 30 K. The irregular spectral changes were interpreted on the basis of the temperature dependence of the linewidth as well as possible hole‐burning phenomena.

Time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene was used to monitor physical changes in the membranes of guinea pig alveolar macrophages following stimulation by N-formyl peptides (either N-formylmethionylphenylalanine (FMP) or N-formyl methionyl leucylphenylalanine (FMLP)) and concanavalin A. The anisotropy of diphenylhexatriene in macrophages showed a dependence on stimulation both in the rate of decay and in the value of anisotropy at infinite time. Subtle differences were observed between the effect of concanavalin A and FMLP on the membrane lipid fluidity as detected by fluorescence anisotropy. Concanavalin A stimulation of macrophages decreased the value of the anisotropy at infinite times in the range of 0–20 °C and increased the value at 25–40 °C; and at all temperatures it decreased the rate of decay of anisotropy. At temperatures below 25 °C, the response to FMLP was similar to concanavalin A, but above 25 °C, FMLP only slightly modified the anisotropy decay profile. Another physical parameter, calcium permeability, was examined because Ca+2 fluxes are dependent upon membrane properties. The temperature-dependent profiles of concanavalin A and FMP-stimulated 45Ca+2 efflux from alveolar macrophages were similar. The rate and extent of 45Ca+2 efflux increased from 4 to 22 °C, with no further increases observed up to 37 °C. This pattern correlated well with observed changes in membrane fluidity.

Overview of factors influencing the appearance and disappearance of the triplet state molecule . . . 3 A. Population of the triplet state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 B. Phosphorescence decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 C. Delayed fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 D. Quenching by neighboring molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 E. Influence of diffusion on quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

The luminescent properties of metal-free, tin(IV) and zinc(II) cytochromes c have been used to characterize the interaction of cytochrome c with mitochondria and cytochrome oxidase. Diminution in the fluorescence yields of tin and zinc cytochrome c occur when these derivates bind to cytochrome oxidase or mitochondria. Based upon spectral overlap and quantum yield, the distance between the porphyrin rings of cytochrome a and cytochrome c is estimated according to Forster theory to be in the neighborhood of 3.5 nm. Measurements of the polarized emission of metal-free 'porphyrin' cytochrome c when bound to oriented layers of cytochrome c oxidase indicate that the porphyrin is bound obliquely to the plane of the oxidase layers with an angle of about 70 degrees C from heme plane to membrane plane. It is proposed that these data have significance for elucidation of electron transfer mechanisms.

Molecular motion of metal-free and metal-substituted cytochrome c derivatives was examined using the anisotropy of emissions from the singlet and the triplet states. The anisotropy of fluorescence provides a means to study the motion of cytochrome c in the nanosecond time scale, since the fluorescence lifetime of metal-free cytochrome c is around 10 ns. We find that the anisotropy of fluorescence of metal-free cytochrome c when bound to mitochondria does not decay, but when bound to phospholipids has a small component which decays independently of the rotation of the whole molecule. The use of phosphorescence extends the time scale for study into the millisecond regime, since the lifetime of the excited triplet state of zinc cytochrome c, as measured by triplet-triplet absorption and phosphorescence emission is approximately equal to 9 ms for free zinc cytochrome c and 7 ms for mitochondrial membrane-bound zinc cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane-bound zinc cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 mus and the slow component with relaxation time of approximately equal to 6 ms. The slow component appears to be due to the rotation of the entire mitochondrion, whereas the fast component was interpreted to be due to the rotation of cytochrome c in a cone about a single axis perpendicular to the plane of the membrane surface.