Protein light absorption

Thirty years of research with bacteriorhodopsin has provided answers to many questions about how protons are transported by transmembrane pumps. In this small seven-transmembrane protein, absorption of light by the retinal chromophore Initiates a reaction cycle in which the initial state recovers through multiple conformational changes of the retinal and the protein, and a proton Is translocated stepwise from one side of the membrane to the other. Spectroscopy, extensive use of site-specific mutations, and crystallography have defined the photocycle reactions in atomic detail and provide a step-by-step description of the proton transfers, the transient local and global perturbations in the protein and how they arise, and the energy flow through the system, which add up to the mechanism of the pump. 

The flat nonselective absorption observed by McLaren for clupein may be ascribed with some confidence to light-scattering, in view of its obvious extension to long wavelengths outside the limits of the protein absorption band proper, though confirmation of this explanation is clearly desirable. For a less concentrated solution of very pure clupein it was observed in this laboratory (Beaven et ed., 1950) that the absorption from ca. 3300 A. down to the onset of the short ultraviolet hands was practically negligible and flat. 

Of the twenty amino acids found in proteins, three may be classified as aromatic in character and account for virtually all of the protein absorption of ultraviolet light above 250 nm and for all of the observed luminescence. The molecular structures of the aromatic amino acids are shown in Fig. 1. The parent aromatic molecules of tryptophan, tyrosine and phenylalanine are, respectively, indole, phenol and benzene. The parents with methyl substituents in place of the amino acid side chains have the common names skatole, p-cresol and toluene, respectively. 

Spectrophotometric assay of P700 and light-induced absorption changes were carried out with a Hitachi 556 dual wavelength spectrophotometer(1,2). EPR measurements of Fe-S proteins were performed as described previously(2). 

In the first absorption spectrum of the mushroom phenolase published by Kertesz and Zito (1957b), an anomalously high protein absorption band at 282 m/i = 27.55) and a low, broad shoulder centered about 340 mix were found. This preparation, at a concentration of 13 mg./ml., had a light yellow color and no absorption bands were visible at the longer wavelengths. This spectrum remained identical, as measured carefully in a standard Beckman DU spectrophotometer, after saturation with carbon monoxide and in the presence of substrate in strict anaerobiosis the apoenzyme, deprived of its copper, also conserved unchanged the original spectrum. 

Figure B2.1.7 Transient hole-burned speetra obtained at room temperature with a tetrapyrrole-eontaining light-harvesting protein subunit, the a subunit of C-phyeoeyanin. Top fluoreseenee and absorption speetra of the sample superimposed with die speetnuu of the 80 fs pump pulses used in the experiment, whieh were obtained from an amplified CPM dye laser operating at 620 mn. Bottom absorption-diflferenee speetra obtained at a series of probe time delays.

What molecular architecture couples the absorption of light energy to rapid electron-transfer events, in turn coupling these e transfers to proton translocations so that ATP synthesis is possible Part of the answer to this question lies in the membrane-associated nature of the photosystems. Membrane proteins have been difficult to study due to their insolubility in the usual aqueous solvents employed in protein biochemistry. A major breakthrough occurred in 1984 when Johann Deisenhofer, Hartmut Michel, and Robert Huber reported the first X-ray crystallographic analysis of a membrane protein. To the great benefit of photosynthesis research, this protein was the reaction center from the photosynthetic purple bacterium Rhodopseudomonas viridis. This research earned these three scientists the 1984 Nobel Prize in chemistry. 

It is generally believed that the absorption (and fluorescence excitation) peak at about 400 nm is caused by the neutral form of the chro-mophore, 5-(p-hydroxybenzylidene)imidazolin-4-one, and the one in the 450-500 nm region by the phenol anion of the chromophore that can resonate with the quinoid form, as shown below (R1 and R2 represent peptide chains). However, the emission of light takes place always from the excited anionic form, even if the excitation is done with the neutral form chromophore. This must be due to the protein environment that facilitates the ionization of the phenol group of the chromophore. This is also consistent with the fact that the pACa values of phenols in excited state are in an acidic range, between 3 and 5 (Becker, 1969), thus favoring anionic forms at neutral pH. 

Mnemiopsin is inactivated by the exposure to light over its entire absorption spectral range, and the inactivation cannot be reversed by keeping the inactivated material in the dark, which differs from the specimens of a live animal. The photoinactivation is accompanied by a partial loss of the 435 nm absorption band, which is probably due to the decomposition of the peroxidized coelenterazine in the protein. 

The photoprotein is non-fluorescent. The absorption spectrum of purified photoprotein shows a very small peak at 410 nm, in addition to the protein peak at 280 nm (Fig. 10.2.5). The peak height at 410 nm appears to be proportional to the luminescence activity of the protein. The protein also shows extremely weak absorption peaks at about 497, 550 and 587nm (not shown). These absorption peaks, except the 280 nm peak, might be due to the presence of a chromophore that is functional in the light emission. 

Campbell and Herring (1987) isolated and partially purified a red fluorescent protein from the suborbital light organs of M. niger. The absorption spectrum of this red fluorescent protein had a peak at 612 nm, a shoulder at 555 nm, and a secondary peak at 490 nm. 

Purines absorb only ultraviolet light and they contribute to structural colors (white and silver) in animals. Pterines are generally yellow, orange, or red pigments. Because they are amphoteric molecules, the absorption spectra depend on the pH and present three or two absorption maxima, usually one in the visible region. Sepiapterin has an absorption maximum at 340 nm in O.IM NaOH and at 410 nm in O.IM HCl." Leucopterin has three maxima 240, 285, and 340 nm. Xanthopterin has two 255 and 391 nm. Because they are conjugated with proteins, pterins show bathochromic shifts in vivo. They also present fluorescence when excited with UV light. 

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