Abstract
Carbon monoxide bound to myoglobin and cytochrome c oxidase in separated adult rat heart myocytes has been observed with Fourier transform IR spectroscopy at low temperatures. CO complexes of these two proteins can be spectrally separated through temperature manipulation of the relaxation of the photolyzed systems. Photolyzed carboxymyoglobin relaxes very rapidly above 80 K, whereas the CO photolyzed from cytochrome a3 associates with CuB and relaxes very slowly below 140 K. Cytochrome c oxidase is found to be present in two major molecular forms which we designate alpha and beta. Each form contains an a3Fe and its associated CuB which we observe by their CO complexes. The predominant FeCO band, the alpha form of cytochrome oxidase, is similar to that previously seen in beef heart mitochondria, but with a slightly larger activation enthalpy, delta H = 46 kJ/mol. At least one of the beta forms is similar, but two have not been observed in beef heart mitochondria. Upon photolysis of alpha-FeCO, the alpha-CuCO species is formed. This band splits into two at low temperature. Up to half of the FeCO band area of the intact myocytes is distributed among three or more minor species (beta forms). The beta-FeCO bands all appear to be associated with only one beta-CuCO band which does not split at low temperature. After photo-dissociation of CO, the beta forms relax considerably faster than the alpha form, achieving 50% recombination in 10% of the time required for the alpha form. In a tissue slice from an opossum heart exposed to CO, we observed alpha and beta forms of cytochrome oxidase very similar to those in the rat heart myocytes. The cause of the differences between the alpha and beta forms of the enzyme is unknown, but their possible role in the control of respiration is discussed. Carboxymyoglobin contained within intact rat heart myocytes was very similar to sperm whale carboxymyoglobin, but with a much smaller amount of the lower frequency minor component.
Highlights
We examined a myocyte preparation that was treated with 16 pg of digitonin/mg of protein to disrupt the sarcolemma and toremove much of the cytoplasmic content, including all myoglobin.* The mitochondria were supplemented with 10
Infrared absorbance difference spectra of COsaturated adult rahteart myocytes provide a clear demonstration of differences between myoglobin- and cytochrome c oxidase-CO-binding sitesand the subpopulations of each
When CO is photolyzed from myoglobin, it weakly associates with the surface of the heme pocket [8] and hasan enthalpy of recombination with the iron of 10 kJ/ mol [9] for sperm whale myoglobin. This is much less than the activation enthalpy for CO recombination with the u3Fe in cytochrome oxidase, which was measured at 40 kJ/mol in beef heart mitochondria [7] and 46 kJ/mol in the glyceroldehydrated rat heartmyocytes investigated in thiswork
Summary
Ately in glass vacutainer tubes filled with CO-saturated glycerol, stoppered, and allowed to dehydrate overnight or transferreddirectly. Both myoglobin and cytochrome oxidase FeCO and their photolysis products can be observed by the CO vibrational absorption. At 120 K, the photolyzed CO in cytochrome oxidase forms a very stable coordination bond with the copper, whereas in myoglobin at this temperature, the photolyzed CO rapidly relaxes back to the iron It is possible, through temperaturemanipulation under conditions of dark and light, to isolate spectrally the two types of proteins. The low concentration of cytochrome c oxidase in the aqueous myocyte pellets limited the CO band intensities For this reason, some samples were prepared by dehydrating the myocyte pellet with glycerol, resulting in a severalfold increase in protein concentration.The additional signal strength made temperature of 160 K on a Digilab FTS-14 FT-IR spectrometer. The CuCO band parameters wereobtainedfrom the spectra through graphic estimates at the same four temperatures
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