András Dezső Kaposi

The two spectroscopically different short wavelength protochlorophyllide forms in pea epicotyls are both monomeric

The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634-636, 644 and 655 nm. At room temperature (293 K) the 628 and 634-636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634-636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634-636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.

Protochlorophyllide forms and energy transfer in dark-grown wheat leaves. Studies by conventional and laser excited fluorescence spectroscopy between 10 K–100 K

The fluorescence properties and role in energy transfer of protochlorophyllide (Pchlide) forms were studied in dark-grown wheat leaves by conventional and laser excited high resolution methods in the 10 K–100 K temperature range. The three major spectral bands, with emission maxima at 633, 657 (of highest intensity) and 670 nm as Bands I, II, and III were analyzed and interpreted as the contributions of six different structural forms. Band I is the envelope of three (0,0) emission bands with maxima at 628, 632 and 642 nm. Laser excitation studies in the range of Band II at 10 K reveal the presence of a spectrally close donor band besides the acceptor, Band II. The intensity in Band III originates mostly from being the vibronic satellite of Band II, but contains also a small (0,0) band with absorption maximum at 674 nm. Excitation spectra show that besides the Pchlides with absorption around 650 nm within Band II, another significant population of Band I with absorption around 640 nm is also coupled by energy transfer to the acceptor of Band II. The spectral difference between the two donor forms indicate different dipolar environments. Upon increasing the temperature, the intensity of Band II and its satellite, Band III decrease, while Band I remains unaffected. Band II shows also a broadening towards the blue side at higher temperatures. Both the quenching of fluorescence and the spectral change was explained by a thermally activated formation of a non-fluorescent intermediate state in the excited state of Pchlide acceptors.

Fluorescence line narrowing applied to the study of proteins

Fluorescence line narrowing is a high resolution spectroscopic technique that uses low temperature and laser excitation to optically select specific subpopulations from the inhomogeneously broadened absorption band of the sample. When applied to the study of fluorescent groups in proteins one can obtain vibronically resolved spectra, which can be analyzed to give information on spectral line shapes, vibrational energies of both the ground and excited state molecule, and the inhomogeneous distribution function of the electronic transitions. These parameters reveal information about the chromophoric prosthetic group and the protein matrix and are functions of geometric strains and local electric fields imposed by the protein. Examples of the use of fluorescence line narrowing are discussed in investigations of heme proteins, photosynthetic systems and tryptophan-containing proteins.

Horseradish peroxidase monitored by infrared spectroscopy: effect of temperature, substrate and calcium

Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in the temperature range of 10-300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positions indicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increased hydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. The substrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-ray crystallography results. With removal of Ca, the protein maintained its overall helicity.

Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.

Native and denatured Zn cytochrome c studied by fluorescence line narrowing spectroscopy

Fluorescence Line Narrowing (FLN) spectroscopy was employed to compare the environment around the porphyrin in folded and unfolded Zn-substituted cytochrome c (Zn cyt c). Parameters of the resolved spectra, including the inhomogeneous energy-distribution function, vibrational energy levels, and phonon coupling, were compared for guanidine-denatured Zn cyt c and native Zn cyt c. The spectra of denatured Zn cyt c showed increased broad background and decreased peak resolution when compared to the native protein, indicating that denaturation results in increased phonon coupling. The energy-distribution function for the unfolded protein was fitted to a single Gaussian centered at 17,230 cm-1 with a width of approx. 360 cm-1, which proved to be blue shifted and much wider than that for native Zn cyt c (approx. 65 cm-1). Vibrational frequencies of the ground-state for Zn cyt c were identified and shown to change upon denaturation. Temperature-dependence of the FLN spectra of native Zn cyt c was analyzed and found to have step-like broadening between 40 K and 50 K. Such discontinuous spectral broadening behavior suggests that a discrete conformational change occurs in the protein at these temperatures.

Solvent dependent and independent motions of CO–horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.

Difference in the transport of metal and free-base porphyrins steady-state and time-resolved fluorescence studies

The binding of Mg-mesoporphyrin and mesoporphyrin to the primary binding site of human serum albumin (HSA) has been studied in the absence and in the presence of 1,2-dimyristoyl-sn-glycero-3- phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPC/DMPG) liposomes. The equilibrium constants of Mg-mesoporhyrin IX (MgMP) and mesoporphyrin IX (MP) for association to HSA were 1.7 . 10(7) (M-1) and 2.5 . 10(7) (M-1), respectively. The association constants for binding to the liposomes were: 1.5 . 10(4) (M-1) for MgMP, and 3.2 . 10(4) (M-1) for MP. The smaller value for the association constants of the MgMP relative to MP in both processes are interpreted as the effect of an out of plane position of Mg2+ and possible ligand co-ordination. HSA added to the liposomes with incorporated porphyrins results in the redistribution of bound molecules. For the MgMP this distribution can be interpreted by the competition of two independent binding processes, while the binding kinetics of MP significantly deviates from this model. The difference is explained by supposing a specific interaction between HSA and the liposomes in case of the free-base MP

Fluorescence line narrowed spectra of Zn and metal-free cytochrome c

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.

Location of Mesoporphyrin in Liposomes Determined by Site-Selective Fluorescence Spectroscopy

Binding of photosensitizers to target cells is a crucial step during the photodynamic effect. Sensitizer distribution is a good indication of whether the chemical is a good candidate for perturbing cell membrane integrity. Hence, the photophysical properties of porphyrinoid sensitizers in microheterogeneous systems such as liposomes are of outstanding interest. Here we present a site-selective fluorescence study of liposome systems. Monocomponent, small unilamellar vesicles formed of different phosphatidylcholines with incorporated mesoporphyrin were investigated. The size distribution of liposomes was measured by dynamic light scattering after each step of the experiment. On the basis of fluorescence line narrowing spectra of mesoporphyrin, the inhomogeneous distribution function was determined in order to characterize the photosensitizer location. The dual character of the functions revealed two different locations. Decomposition of the inhomogeneous distribution functions into Gaussians and the analysis of the fit results suggest that one of the locations for mesoporphyrin is between the two lipid layers, and the other one is between the hydrocarbon chains of the lipid molecules.