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, band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin

PROTEIN CONFORMATION MONITORED BY ENERGY-SELECTIVE OPTICAL SPECTROSCOPY

Fluctuations in the polypeptide chain lead to disorder in proteins and to a distribution in the parameters that regulate their functions. Using low temperature (to reduce the fluctuations) and narrow-band lasers (to select one substate among the many forms), high-resolution absorption and fluorescence spectra for chromophores in proteins can be obtained. These spectra reveal information on the kind and extent of disorder in proteins and allow for the determination of the vibrational energies of both ground and excited state molecules, true inhomogeneous spectral width, and kinetic studies of individual protein substates

Consequences of inhomogeneous broadening on fluorescence line narrowing spectra

Fluorescence line narrowing is a sensitive tool to monitor subtle changes in protein conformation. We restudied the consequences of inhomogeneous broadening on the observed spectra. Vibronic absorption bands of systems with weak electron-phonon coupling can be described by a lorentzian function with width of the order of a few centimeters. For an ensemble of molecules with closely spaced electronic transitions, a single laser frequency will excite many molecules through the broad base ("tail") of the lorentzian function. This, along with excitation of low-frequency modes, contributes to the unresolved background observed in line-narrowed spectra. Examples are shown for Zn cytochrome c, a fluorescent derivative of Fe cytochrome c. Spectra are compared for the protein in two solvents: glycerol/water or trehalose. Two types of cytochrome, from horse and yeast, are also compared

In situ detection of ALA-stimulated porphyrin metabolic products inEscherichia coli B by fluorescence line narrowing spectroscopy

In a recent work [Photochem. Photobiol. B: Biol. 50 (1999) 8] the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on delta -aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation. In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques. Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed. We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium. Incorporation of Zn ions originating from the glassware could also be supposed. In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins. The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins. The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population. (C) 2001 Elsevier Science B.V. All rights reserved

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. (C) 1998 Elsevier Science B.V. All rights reserved

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. (C) 1998 Elsevier Science B.V. All rights reserved

The surgical technique and the age of the horse both influence the outcome of mosaicplasty in a cadaver equine stifle model

Six pieces of grafts, 6.5 mm in diameter, 20 mm in length, were taken from each of 170 cadaver hindlimbs, using the cranial surface of the medial femoral trochlea for harvesting. The age of the horses varied between 4 months and 23 years. 30 limbs under the age of 12 years were selected for transplantation. Three of six grafts were transplanted into the medial femoral condyle using different combinations of tunnel depth and dilation. With ageing, a significant decline in transplantability was detected. In general, mosaicplasty cannot be recommended in horses above ii years. Based on a previous clinical case (Bodo et al., 2000), a good surface alignment was indeed achieved with a combination of graft length drilling and dilation in most cases. However, the occasional entrapment of cartilage debris under the graft prevented perfect alignment in the present cadaver study in 27% of the grafts transplanted in this manner. Since the protrusion of grafts never exceeded 1.5 mm, we conclude that drilling 3-5 mm deeper than graft length with graft length deep dilation can avoid disadvantageous protrusion of the transplanted hyaline cartilage caps, achieving bone decompression at the same time

Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi, transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix. (C) 2002 Wiley Periodicals, Inc

Water channel of horseradish peroxidase studied by the charge-transfer absorption band of ferric heme

The heme of horseradish peroxidase is buried in the protein, but a channel from the protein surface connects the aqueous solution to the heme site. Ferric horseradish peroxidase has an absorption band at 640 nm that is attributed to a charge-transfer (CT) transition between the a(2u) HOMO of T electrons of the porphyrin ring and the d(xy)/d(yz) orbital of the ferric ion. Because the water channel extends to the Fe, it seems likely that the CT band will be sensitive to the hydration of the protein. To study this premise, the protein was incorporated into trehalose/sucrose glasses and the hydration of the sugar glasses was varied. Absorption spectra of HRP in sugar glasses and in glycerol/water were taken in the range 10-300 K. The CT absorption band shows vibronic fine structure. The peak positions are the same in hydrated sugar and glycerol/water but the peak positions change in desiccated sugar glass. The data suggest that in hydrated, but not desiccated, sugar glass, water is retained in the heme pocket. Binding of the competitive inhibitor benzohydroxamic acid to the protein increases the CT absorption and resolution. The effect of benzohydroxamic acid on the Fe as calculated using a combination of density functional theory and molecular mechanics is to stabilize the spin state 3/2 with respect to 5/2. At low temperature the widths of the lines in the CT band are narrower for the protein in glycerol/water (glass transition at similar to150 K) than in trehalose/sucrose (glass formation at 65 degreesC). This indicates that the CT band is inhomogeneously broadened and sensitive to the solvent. The spectral narrowing of the CT absorption occurs as the temperature decreases over the temperature range studied. Water, as indicated by the OH stretch, also shifts in this range. The findings are discussed in terms of how buried water and nearby charges can modulate the activity of the heme

Carbonmonoxy horseradish peroxidase as a function of pH and substrate: Influence of local electric fields on the optical and infrared spectra

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6.0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrational stretch frequency is at 1903 cm(-1). Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of similar to 320 cm(-1) in the Q(0,0) absorption band that is clearly evident at (CO) to a higher frequency (1936 cm(-1)). The splitting of the Q(0,0) band and the shifts in the IR spectra are both consistent with changes in the local electric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the distal histidine, although other effects are also considered. The larger effect on the Q(0,0) band with substrate at low pH and the shift of nu (CO) at high pH can be rationalized by the directionality of the field and the orientation dependence of dipolar interactions