Complex formation between chlorophyll a and cytochrome c: surface properties at the air‐water interface

Abstract

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (n), surface potential ( A V) and ['4C]cytochrome c surface-concentration (r) isotherms were measured versus molecular area (a) in mixed films. Compared to the successive-addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN . m-'. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression-expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of ['4C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir-Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a-cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energytransfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.The morphological basis of biological functions in membranes is very difficult to determine, due to the complexity, with regard to both the organization and nature of membrane components. In photosynthetic membranes it is known that chlorophyll is associated with membrane proteins [l -31. Such molecular associations can only be extracted and purified with the use of detergents, which can be reasonably assumed to affect their integrity. Therefore, synthetic approaches have been considered in order to reconstitute, gradually, the biological membrane functions. The monolayer technique is widely used, since it allows a fine control of molecular orientation and it deals with concentrations similar to those found in natural membranes. Furthermore, this technique permits the study of the effects of different parameters, such as temperature, pH and ionic strength, either in succession or simultaneously. Finally, monolayer methods can be used to investigate mixed films with pure components, alone or mixed in any molar ratio. Despite such advantages, it is not easy to investigate chlorophyll-protein complexes using monolayer methods, since the integral proteins are highly hydrophobic Correspondence to R. M. Leblanc, Centre de recherche en photobiophysique, Universite du Quebec a Trois-Rivieres, C.P. 500, TroisRivikres, Quebec...

Quantitative analysis of molecular orientation in chlorophyll a Langmuir monolayer: a polarized visible reflection spectroscopic study

“…The structure and aggregation states of chlorophyll a films have been investigated for many years (Jacobs et al, 1954;Kreutz, 1968;de B.Costa et al, 1972;Le Brech et al, 1974;Leblanc et al, 1974;Chapados and Leblanc, 1977;Aghion and Leblanc, 1978;Chapados et al, 1980;Dodelet et al, 1980;Jones et al, 1980;Aghion et al, 1981;Lawrence et al, 1981;Frackowiak et al, 1983;Chapados and Leblanc, 1983;Lawrence et al, 1984;Desormeaux and Leblanc, 1985;Diarra et al, 1986;Bardwell and Dignam, 1987;Lamarche et al, 1991;Desormeaux et al, 1993) because of the potential utility for solar energy conversion into electrochemical energy. However, the structure and molecular orientation in Langmuir monolayers of chlorophyll a has not been fully understood yet.…”

Importance of the Spreading Solvent Evaporation Time in Langmuir Monolayers

“…During the years, the spreading solvent choices for researchers focusing on surface chemistry at the air−water interface has ranged from pure solvents such as chloroform, toluene, , and benzene , to solvent mixtures such as chloroform or hexane:methanol or hexane:ethanol (5:1 or 9:1, v/v) depending on the characteristics of the amphiphilic molecule under study. The rationale to choose a spreading solvent is its immiscibility with the water subphase and a fairly fast evaporation rate .…”