Photophysics and Photochemistry of Horseradish Peroxidase A2 upon Ultraviolet Illumination
Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H^sub 2^O^sub 2^ to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein’s enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T^sub m^ values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme’s Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein’s fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.
Peroxidases are heme-containing enzymes that reduce H^sub 2^O^sub 2^ to oxidize a wide variety of organic and inorganic compounds. The heme prosthetic group, appointed “ferriprotoporphyrin IX”, allows electron transfer between reductant and oxidant substrates (1). During catalysis in plant peroxidases a porphyrin cation ? radical is formed (2), and in yeast cytochrome c peroxidase a trypiophan radical plays the equivalent role (3,4). In both cases in the first reaction step the iron ion becomes hexacoordinated to an oxygen atom (Fe^sup IV^=O). In classical plant peroxidases tryptophan is uniquely represented and is highly conserved, located in a surface loop connecting two helices. The indole ring is directed toward the core of the protein lying above the plane defined by the heme group at an average distance of 16-18 [Angstrom] (5-8) (Fig. 1 A). Trp fluorescence in classical plant peroxidases is highly quenched in the native state due to energy transfer from the excited state of tryptophan to the heme group (5,9,10). Quantum yields are 100 orders of magnitude lower than that of tryptophan free in solution. Ferriprotoporphyrin also displays a visible absorption spectrum.
The most important UV light absorbers in proteins are the aromatic residues, Trp, Tyr, and Phe along with cystine and His (11). It is known that tryptophan and tyrosine radicals can be induced by ultraviolet (UV) illumination (12-14). Heme groups are also excellent UV absorbers. UV illumination might in some cases induce enzyme inactivation ( 15-18). The mechanisms of inactivation are believed to be due to ionization of aromatic residues associated with an electron transfer mechanism and radical formation together with disruption of disulphide bridges (19-23). Earlier studies confirm that the major reaction after UV excitation of enzymes is phototonization (24). The excited tryplophan after photoionization gives TrpH^sup +^, and electron is ejected from the molecule. In heme proteins, the TrpH^sup +^ might reduce the ferrous heme (25). Porphyrins are a major source of free radicals and singlet oxygen and can induce photobleaching in fluorescent molecules (26). Several studies are reported on photoinactivation of catalase, a tetrameric heme-containing enzyme. Catalase degrades H^sub 2^O^sub 2^ to oxygen and water and represents an important part of the antioxidative system in cells. Blue light (380-500 nm) illumination of sunflower catalase causes partial heme destruction, and oxidation of histidine, present in the catalytic center of catalase, perhaps is an early event in photoinactivation (27). UV irradiation (365 nm) of bovine liver catalase leads to the formation of calalytically inactive compounds III (oxyferrous catalase) and II (catalase FeIV) (28). UV C irradiation of flavocytochrome b^sub 2^, which houses a heme prosthetic group, results in enzyme inactivation, where Tip and Tyr residues as well as the heme group are modified (25). Oxygen presence offers protection of tryptophan against UV-induced damage due to quenching (11), whereas the heme group can act as a photosensitizer thus generating harmful singlet oxygen (29).
This work presents a detailed spectroscopic analysis of UV-induced changes in secondary structure, enzymatic activity, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption of horseradish peroxidase A2 (HRPA2). The catalytic activity, conformational changes at the secondary structure level, protein fluorescence, and heme absorption were monitored after Trp 296-nm UV illumination. A streak camera study on the effects of UV illumination time on the distribution of the two shortest fluorescence lifetimes of the single endogenous aromatic residue Trp in HRPA2, at pH 4, is presented. We also highlight the outstanding value of streak camera technology in following ultrafast processes and show how streak camera data can be correlated with protein structural changes. Also, we hereby present the effects of continuous illumination of the heme group at 403 nm on Trp fluorescence emission and on heme absorption bands.
Chemicals and sample preparation
Horseradish peroxidase was obtained from Biozyme Laboratories (Blaenavon. UK) (labeled HRP-5) as lyophilized salt-free powder and used without further purification. HRP-S corresponds to HRPA2 according to the nomenclature of Shannon et al. (30) and data reported by Hiner et al. (31). The degree of purity was checked by sodium dodecylsulfaie-polyacrylamide gel electrophoresis, and a single band was observed when stained with Coomassie. HRPA2 concentrations were determined considering the molar extinction coefficient ?^sub 4030n^, = 100 mM^sup -1^cm^sup -1^ (32). Protein solutions used for spectroscopic studies were dissolved in buffer made with Milli-Q water. All salts were of analytical grade. Acetate buffer was used for pH 4. Buffer concentration was always 25 mM.
Extinction coefficient as a function of wavelength of HRPA2 and hematin
The absorbance from 250 nm to 700 nm of a horseradish peroxidase solution prepared as mentioned above was measured on a Thermo Electron (Waltham, MA) UV1 spectrophotometer and compared with a study performed by Du et al. (33) on the absorption of hematin in acetic acid.
Tryptophan irradiation-steady-state fluorescence
The enzyme solution (3 mL of a 5 µM protein solution) was continuously irradiated for different time periods at 296 nm using a 75 W Xenon are lamp from an RTC 2000 PTI (Photon Technology International. Birmingham, NJ) spectrometer provided with a monochromator. Excitation and emission slit widths were set to 6 nm. Tryptophan fluorescence was monitored at 350 nm (excitation spectra) and excited at 296 nm (emission spectra). Temperature in the cell, a quartz cuvette ( 1 -cm path length) was controlled using a Peltier element. The sample was continuously slined at 650 rpm to maintain the homogeneity of the solution, and the temperature was kept constant at 20°C. Line voltage was controlled and maintained at 4 V. thus avoiding fluctuations deriving from the power coming from the electrical outlet.
Activity measurements, steady-state fluorescence spectra, far-UV circular dichroism (CD) spectra, and denaturation curves after ellipticity at 223 nm were determined for samples irradiated for different time periods. Control samples, without irradiation, underwent the same treatment as the exposed ones.
Activity measurements
HRPA2 activity was measured at room temperature using 50 mM guaiacol in 25 mM acetate buffer (pH 4) and 4.4 mM H^sub 2^O^sub 2^. The reaction was followed for 1 min by reading the increase in absorbance at 470 nm. The extinction coefficient of the oxidation product, ?^sub 470nm^ = 26.6 mM^sup -1^cm^sup -1^, was used to calculate initial velocities.
Far-UV CD measurements
CD measurements were carried out using a Jasco (Tokyo, Japan) spectropolarimeter, model J-715. The ellipticity values were obtained in mdegrees directly from the instrument and converted to the mean residue ellipticity ?^sub MRW^ as previously stated (8). The far-UV CD spectra were measured using a rectangular quartz cell of 1-mm path length. Each spectrum was an average of six scans between 300 and 200 nm. The resultant ellipticities of the HRPA2 solutions were calculated by subtracting the elliplicity of the buffer solution. The wavelength of 223 nm was used to monitor thermal denaluration in the far-UV CD range. Temperature scans were carried out in the temperature range 293-358 K using a Pettier element (irradiated samples] or a thermostaled cuvette by means of u circulating water bath, and a temperature probe was immersed in the protein (dark control samples). The experimental parameters were as follows: 1-nm bandwidth, 0.2-K step resolution, 2-s response time, and scanning rates 1.5 (irradiated samples) and 2.6 K min^sup -1^ (control).
HRPA2 home irradiation studies
Irradiation and steady-state fluorescence
A total of 3 mL of a 4 µM protein solution was continuously irradiated for 30 h at 403 nm using a 75-W Xenon are lamp from a RTC 2000 PTI (Photon Technology International) spectrometer provided with a monochromaior (slits width 6 nm). Temperature in the cell, a quartz cuvette (1-cm path length) was controlled using a Peltier element and was kepi constant at 298 K. The sample was continuously stirred at 650 rpm to maintain the homogeneity of the solution. Line voltage was controlled and maintained at 4 V, thus avoiding fluctuations deriving from the power coming from the electrical outlet. Before 403 nm irradiation and at specified times during the irradiation, tryptophan fluorescence emission intensity at 350 nm upon 296-nm excitation was acquired.
Irradiation and absorption measurements
In another experiment, absorption by the protein solution was monitored before and after irradiation with 403-nm light for 26 h. The sample was irradiated using the same conditions as above. Measurements were performed on a Thermo Electron UV1 spectrophoiometer.
To model excited state processes or to unravel heterogeneity in the distribution of fluorophores, experiments under a variety of conditions can be performed. One tun change experimental parameters such as excitation and emission wavelengths, pH, quencher concentration, timescale, temperature, orientation of excitation, and emission polarizers. Finally, a multi-dimensional fluorescence decay surface is obtained. From the separate analyses of the individual decay traces, a model can be deduced. The appropriateness of the model is checked by verifying the consistency of the parameter values obtained from each decay curve analysis. However, the parameter estimates resulting from the various single decay curve analyses may suffer from u large uncertainty so that the model building becomes difficult. It has to be realized that many parameters appear in a nonlinear way in the model function and that in most cases the functions within the model are nonorthogonal. It has been suggested Io perform a simultaneous analysts of related decay traces, i.e., of the fluorescence decay surface, by linking the common parameters. The merits of this global analysis approach have been emphasized and used broadly within the scientific community (34). Global analysis of fluorescence lifetime data can be used to obtain an accurate lit of multi-exponential fluorescence decays. Global analysis algorithms simultaneously tit multiple measurements acquired under different experimental conditions to achieve higher accuracy.