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Squid rhodopsin (lambda(max) 493 mmicro)-like vertebrate rhodopsins-contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (lambda(max) 500 mmicro) near neutrality, yellow (lambda(max) 380 mmicro) in alkaline solution. The two forms-acid and alkaline metarhodopsin-are interconverted according to the equation, Alkaline metarhodopsin + H(+) right harpoon over left harpoonacid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.

Rhodopsin, the pigment of the retinal rods, can be bleached either by light or by high temperature. Earlier work had shown that when white light is used the bleaching rate does not depend on temperature, and so must be independent of the internal energy of the molecule. On the other hand thermal bleaching in the dark has a high temperature dependence from which one can calculate that the reaction has an apparent activation energy of 44 kg. cal. per mole. It has now been shown that the bleaching rate of rhodopsin becomes temperature-dependent in red light, indicating that light and heat cooperate in activating the molecule. Apparently thermal energy is needed for bleaching at long wave lengths where the quanta are not sufficiently energy-rich to bring about bleaching by themselves. The temperature dependence appears at 590 mmicro. This is the longest wave length at which bleaching by light proceeds without thermal activation, and corresponds to a quantum energy of 48.5 kg. cal. per mole. This value of the minimum energy to bleach rhodopsin by light alone is in agreement with the activation energy of thermal bleaching in the dark. At wave lengths between 590 and 750 mmicro, the longest wave length at which the bleaching rate was fast enough to study, the sum of the quantum energy and of the activation energy calculated from the temperature coefficients remains between 44 and 48.5 kg. cal. This result shows that in red light the energy deficit of the quanta can be made up by a contribution of thermal energy from the internal degrees of freedom of the rhodopsin molecule. The absorption spectrum of rhodopsin, which is not markedly temperature-dependent at shorter wave lengths, also becomes temperature-dependent in red light of wave lengths longer than about 570 to 590 mmicro. The temperature dependence of the bleaching rate is at least partly accounted for by the temperature coefficient of absorption. There is some evidence that the temperature coefficient of bleaching is somewhat greater than the temperature coefficient of absorption at wave lengths longer than 590 mmicro;. This means that the thermal energy of the molecule is a more critical factor in bleaching than in absorption. It shows that some of the molecules which absorb energy-deficient quanta of red light are unable to supply the thermal component of the activation energy needed for bleaching, so bringing about a fall in the quantum efficiency. The experiments show that there is a gradual transition between the activation of rhodopsin by light and the activation by internal energy. It is suggested that energy can move freely between the prosthetic group and the protein moiety of the molecule. In this way a part of the large amount of energy in the internal degrees of freedom of rhodopsin could become available to assist in thermal activation. Assuming that the minimum energy required for bleaching is 48.5 kg. cal., an equation familiar in the study of unimolecular reaction has been used to estimate the number of internal degrees of freedom, n, involved in supplying the thermal component of the activation energy when rhodopsin is bleached in red light. It was found that n increases from 2 at 590 mmicro to a minimum value of 15 at 750 mmicro. One wonders what value n has at 1050 mmicro, where vision still persists, and where rhodopsin molecules may supply some 16 kg. cal. of thermal energy per mole in order to make up for the energy deficit of the quanta.