Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method

Abstract

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.

Molecular Organization and Dynamics in Bacteriorhodopsin-Rich Reconstituted Membranes: Discrimination of Lipid Environments by the Oxygen Transport Parameter Using a Pulse ESR Spin-Labeling Technique

“…A model for the mechanism of oxygen transport in the membrane has been proposed in which molecular oxygen enters transient small vacant pockets created by gauche-trans isomerization of alkyl chains or by the structural nonconformability of neighboring lipids (mismatch in surface topography of molecules when two molecules are placed side by side in the membrane), and oxygen molecules jump from one pocket to an adjacent one or move along with the movement of the pocket itself (Trauble, 1971;Pace & Chan, 1982;Subczynski et al, 1991a;Haines & Liebovitch, 1993). This mechanism for the diffusion of small molecules in the membrane is quite different from that for the lateral diffusion of proteins and lipids in the membrane (Pace & Chan, 1982).…”

The aqueous reference for ESR oximetry

“…The use of nitroxide ESR to measure oxygen concentration has provided considerable information about the distribution of oxygen in a variety of heterogeneous biological environments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The method depends on the change in the electron spin relaxation induced in nitroxide radicals by collisional encounters with oxygen.…”

Organization of lipids in fiber-cell plasma membranes of the eye lens

“…Membrane proteins are nearly impermeable to oxygen [97, 98]. Thus, the total effective oxygen permeability coefficient across the intact fiber cell plasma membrane is equal to the oxygen permeability coefficient evaluated for the lipid bilayer portion of the membrane, decreased by a factor proportional to the surface area of the lipid bilayer portion, divided by the surface area of the entire membrane.…”