Phototoxicity and photoinactivation of blebbistatin in UV and visible light

Blebbistatin was recently identified as a selective, cell-permeant inhibitor of myosin II. Because blebbistatin is likely to be used extensively with fluorescence imaging in studies of cytoskeletal dynamics, its compatibility with common excitation wavelengths was examined. Illumination of blebbistatin-treated bovine aortic endothelial cells at 365 and 450-490 nm, but not 510-560 or 590-650 nm, caused dose-dependent cell death. Illumination of blebbistatin alone at 365 and 450-490 nm changed its absorption and emission spectra, but the resultant compounds were not toxic. In addition, photoreacted blebbistatin no longer disrupted myosin distribution in cells, indicating loss of pharmacological activity. Fluorescence microscopy showed that upon illumination, blebbistatin became bound to cells and to protein-coated glass, suggesting that toxicity may arise from light-induced reaction of blebbistatin with cell proteins. Blebbistatin should be used only with careful consideration of these photochemical effects.     

Light-induced movement of magnesium ions in intact chloroplasts. Spectroscopic determination with Eriochrome Blue SE

The metallochromic indicator Eriochrome Blue SE was used to measure light-induced internal movement of Mg²⁺ in intact chloroplasts. By dual-wavelength spectroscopy (measuring wavelength 554 nm, reference 592 nm) a light-induced, dark-reversible absorbance increase of Eriochrome Blue in samples of isolated intact chloroplasts was observed. The light/dark difference spectrum of Eriochrome Blue between 550 and 590 nm (reference wavelength 562 nm) indicated that this absorbance increase was caused by an increased concentration of free Mg²⁺ in a neutral or slightly alkaline chloroplast compartment.

The signal was seen only with intact, but not with broken, envelope-free chloroplasts, which had lost most of their divalent cations. This is interpreted to show that the indicator responds to an increase of Mg²⁺ concentration in the chloroplast stroma, which represents an efflux of Mg²⁺ from the intra-thylakoid space caused by light-dependent proton pumping.

As calculated from corrected values of the absorbance increase of Eriochrome Blue, the light-induced internal release of Mg²⁺ was close to 100 nequiv per mg chlorophyll at pH 7.6 and 250 nequiv at pH 7.1. This corresponds to a light-dependent increase in the concentration of free Mg²⁺ in the stroma of about 2 and 5 mM, respectively.     

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides: an analysis and interpretation of the band shifts.

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20% of the total carotenoid content, is responsible for the light-induced absorbance changes. The 'remaining' pool absorbs at wavelengths which were about 5 nm lower than those at which the 'changing' pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields. The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 +/- 6 mV per nm shift.     

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides. An analysis and interpretation of the band shifts

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20 % of the total carotenoid content, is responsible for the light-induced absorbance changes. The ‘remaining’ pool absorbs at wavelengths which were about 5 nm lower than those at which the ‘changing’ pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields.

The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 ± 6 mV per nm shift.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor.

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550-650 and 700-950nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as deltaav/deltaah, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm , and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the "special pair" that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the "light-dark" difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

Light and dark adaptation of halorhodopsin

Dark incubation of envelope vesicles derived from a strain of Halobacterium halobium that lacks bacteriorhodopsin but contains halorhodopsin and a third rhodopsin-like pigment caused a decrease in the flash yield [the amplitude of a transient absorbance change of flash reactive component(s) by flash] of halorhodopsin but not the rhodopsin-like pigment. The flash yield decreased to reach a low steady level after incubation for about 4 days in the dark. The flash yield of halorhodopsin at any stage of dark incubation was increased by actinic illumination of the vesicles. The flash yield at 490 nm (absorbance increase) was found to be approximately proportional to that at 590 nm (absorbance decrease). These results indicate that halorhodopsin in the envelope vesicles has two forms, dark and light adapted, and that the halorhodopsin phototransient absorbing at 490 nm is originated from the light-adapted form. A difference spectrum between these two forms of halorhodopsin shows that the light-adapted halorhodopsin was red-shifted from the dark-adapted form. The light-induced membrane potential was measured by tetraphenylphosphonium uptake. The uptake by the dark-adapted vesicles was slower than that by the light-adapted vesicles, suggesting that only the light-adapted halorhodopsin has ion-transporting activity.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550–650 and 700–950 nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as Δa_v/Δa_h, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810 nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm, and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the “special pair” that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the “light-dark” difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

Mechanism of photochemical oxidation of bacterioviridin]

The mechanism of bacterioviridin photochemical oxidation has been studied by the methods of ESR, flash-photolysis and low-temperature spectrophotometry. ESR spectrum of pigment cation-radical, a singlet line with H=11 G, g = 2.0027, has been recorded. The bands with maxima at 370, 470, 525, 590, 840 nm correspond to bacterioviridin cation -- radical in the absorption spectra. When -- benzoquinone is used as an electron acceptor with excitation light 640 nm the product of bacterioviridin irreversible oxidation is formed with the absorption band maximum 760 nm and absorption between 350 and 370 nm. It is suggested that this product is of double-oxidized non-radical nature and the mechanism of its formation through oxidation of the pigment cation-radical is discussed. The regeneration reaction of double-oxidized bacterioviridin up to cation-radical form in the presence of triphenylamine as a reducing agent has been carried out. The rate constants of cation-radical decay in the dark and desactivation of triplet state have the following values: K1=(1,64+/-0,15)-10(3) sec-1, K2=(13+/-2,0)-10(3) sec-1 correspondingly. The activation energy of the radical decay in the dark is Eact =(13,2-0,5) kcal/mole.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

Light-induced absorption changes in Photosystem I at low temperatures

Light-induced absorption changes associated with the primary photochemical reaction and dark relaxation in Photosystem I were measured at various low temperatures. A possible temperature-dependent long-range electron tunneling process was suggested to account for the unique temperature dependence of the dark decay process. The kinetics of the light-induced absorption changes are in good agreement with the light-induced EPR changes reported earlier (Ke, B., Sugahara, K., Shaw, E.R., Hansen, R. E., Hamilton, W. D. and Beinert, H. (1974) Biochim. Biophys. Acta 368, 401–408) for the same Photosystem I subchloroplast fragments at comparable temperatures.All absorption changes between 400 and 725 nm at 86 °K have identical kinetics. The light-minus-dark difference spectrum is very similar to that of P-700 at room temperature, with an additional prominent positive change at 690 nm. Possible contributions by P-430 to the blue and red spectral changes were discussed.It was demonstrated that the intensity of the measuring beam has a drastic effect on the light-induced absorption changes of Photosystem I at low temperatures. Various pretreatments of the Photosystem I fragments such as those that photochemically (or chemically) oxidize the primary donor or photoreduce the primary acceptor abolish the subsequent photochemical reaction. Continuous illumination of the Photosystem I fragments before and during freezing has the same effect.In the temperature range of ?20 to ?60 °C, an unusual counter absorption change as well as a counter EPR change were observed.     

Light-induced absorbance changes of carotenoids in brown algae

Light-induced absorbance changes were studied for brown algae with 23 species and a pronounced absorbance change around 563 nm was found in all algae examined. 3-(3,4-dichlorophenyl)-1,1-dimethylurea and gramicidin J suppressed the initial rate and the magnitude of the absorbance change. Carbonylcyanidem-chlorophenylhydrazone did not affect the initial rate but decreased the maximum level of the change. All thalli and the chloroplasts tested had an absorption band at around 540 nm due to fucoxanthin which accounted for about 70–90% of the total carotenoids in brown algae. It is proposed that the 563 nm-change is caused by the red shift of fucoxanthin responding to the light-induced change in the membrane potential of the thylakoid system.     

Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase

Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase. This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm. Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores. A histidine ligand probably is also present. Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation. Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate [Cu(II)-S(Cys)N(His)] sites or with inequivalent and uncoupled cysteine ligands in the same site. The former possibility is considered to be more likely.     

Feulgen type staining of mammalian tissues with Dahlia-SO2.

The use of the basic dye, Dahlia, which belongs to triphenylmethane group but without a primary amino group in its molecule has been described as useful in the staining of aldehyde groups of acid hydrolysed DNA in tissue sections following the conventional Feulgen procedure. Dahlia-SO2 prepared with sodium hydrosulphite is highly suitable when used at pH 4-0 to 5-0. The absorption characteristics of the stained nuclei indicate on the peak of maximum absorption at 560 nm, whereas, that of the aqueous dye solution is at 590 nm.     

Oxidation-reduction potential dependence of low-temperature photoreactions of chloroplast Photosystem II

The light-induced free-radical signal of Photosystem II (observed after illumination at 77 °K) has been studied in chloroplasts as a function of the oxidation-reduction potential established prior to freezing. The intensity of the light-induced signal is unchanged in the potential region of +590 mV to +760 mV. At higher potential (+850 mV), there is a 30% decrease in signal intensity. The light-induced signal decreases to zero in the low-potential region, with a midpoint potential of +475 mV. These results are considered in terms of a Photosystem II reaction-center complex in which the light-induced free-radical signal arises from the oxidized form of the reaction-center chlorophyll, and this chlorophyll molecule is capable of being reduced at liquid-nitrogen temperature by a secondary electron donor which has a midpoint oxidation-reduction potential of +475 mV.     

Characterization of a soluble catalase-peroxidase hemoprotein b-590, previously identified as 'cytochrome alpha 1' from Bradyrhizobium japonicum bacteroids.

The cytochrome "a1" or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified. The water-soluble native hemoprotein has an Mr of 136,000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b-590 (Escherichia coli; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO. It has catalase and peroxidase activities, again resembling the E. coli b-590. Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish peroxidase reacts with oxygen to form such a complex. The hemoprotein, which we name hemoprotein b-590 (Bradyrhizobium japonicum), may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species. The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.     

Haemoprotein b-590 (Escherichia coli), a reducible catalase and peroxidase: evidence for its close relationship to hydroperoxidase I and a 'cytochrome a1b' preparation

A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The Mr of the native protein, determined by gel filtration, was 331,000 although a minor, smaller species with a Mr of 188,000 was also detected; both had catalase activities. Based on the subunit Mr, determined from SDS gel electrophoresis to be 75,000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4.4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A405/A280 ratio never exceeded 0.27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the gamma, beta, and alpha bands were at 441, 559 and 590 nm respectively, the alpha-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent gamma-band at 426 nm and caused significant shifts of the alpha and beta bands to shorter (574 and 545 nm) wavelengths. The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: Vmax was 2.3 X 10(6) mol H2O2 (mol haem)-1 min-1 and the Km was 11 mM. At 10 mM-H2O2 the first order rate constant was 0.3 X 10(7) M-1 s-1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3',6-trichloroindophenol as substrates; for the latter substrate, the Km was 0.18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).     

Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.     

Transient optical spectroscopy of single crystals of the reaction center from Rhodobacter sphaeroides wild-type 2.4.1

The photoactivity of the crystallized reaction centers from Rhodobacter sphaeroides wild-type strain 2.4.1 has been examined by light-induced absorption spectral changes associated with charge separation and triplet state formation in the reaction center. Upon excitation of a crystal at ambient redox potential, the primary donor 865 nm band bleaches reversibly. The kinetics of its recovery were found to be biphasic with rate constants 11.5 +/- 1.3 s-1 and 0.9 +/- 0.4 s-1 which correspond to lifetimes of 87.0 +/- 9.0 ms and 1.0 +/- 0.7 s, respectively. The ratio of the fast-to-slow component preexponential terms was 3.5 +/- 1.1 suggesting that the majority (78.9 +/- 13.0%) of the reaction centers in the crystals lack the secondary quinone, QB. The addition of sodium ascorbate to the crystals attenuates the 865 nm absorption change, and gives rise to strong carotenoid triplet-triplet absorption changes at 547 nm. These data indicate that the reaction center-bound carotenoid in the crystals is capable of accepting triplet energy from the primary donor triplet.     

Shifts of the bacteriochlorophyll absorption band at 880 nm in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum]

The redox potential dependency of the light-induced absorption changes of bacteriochlorophyll in the chromatophores and subchromatophore particles from Rhodospirillum rubrum has been studied. The highest values of the absorption changes due to the bleaching of P870 and the blue shift of P800 are observed within the redox potential range of 360--410. At the potential values below 300 mV the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in the subchromatophore particles and to longer wavelengths in the chromatophores. Redox titration revealed that the red and blue shifts of 880 nm bacteriochlorophyll band are caused by the functioning of a non-identified component (X) which has an oxidation -- reduction midpoint potential close to 340 mV (n = 1) within the pH range of 6,0--7,6. The Em for this component decreases by 60 mV/pH unit within the pH range of 7.6--9,2. The results obtained suggest that the red shift is due to the transmembrane, while the blue shift -- to the local intramembrane electric field. The generation of both the transmembrane and local intramembrane electric fields apparently depends on redox transitions of the component X.     

Correlation of proton release and electrochromic shifts of the optical spectrum due to oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides

Reaction centers from the Y(L167) mutant of Rhodobacter sphaeroides, containing a highly oxidizing bacteriochlorophyll dimer and a tyrosine residue substituted at Phe L167, were compared to reaction centers from the Y(M) mutant, with a tyrosine at M164, and a quadruple mutant containing a highly oxidizing dimer but no nearby tyrosine residue. Distinctive features in the light-induced optical and EPR spectra showed that the oxidized bacteriochlorophyll dimer was reduced by Tyr L167 in the Y(L167) mutant, resulting in a tyrosyl radical, as has been found for Tyr M164 in the Y(M) mutant. In the Y(L167) mutant, the net proton uptake after formation of the tyrosyl radical and the reduced primary quinone ranged from +0.1 to +0.3 H(+)/reaction center between pH 6 and pH 10, with a dependence that is similar to the quadruple mutant but different than the large proton release observed in the Y(M) mutant. In the light-induced absorption spectrum in the 700-1000 nm region, the Y(L167) mutant exhibited unique changes that can be assigned as arising primarily from an approximately 30 nm blue shift of the dimer absorption band. The optical signals in the Y(L167) mutant were pH dependent, with a pK(a) value of approximately 8.7, indicating that the tyrosyl radical is stabilized at high pH. The results are modeled by assuming that the phenolic proton of Tyr L167 is trapped in the protein after oxidation of the tyrosine, resulting in electrostatic interactions with the tetrapyrroles and nearby residues.