Abstract
The geminate rate constants for CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide rebinding to soybean leghemoglobin and monomeric component II of Glycera dibranchiata hemoglobin were measured at pH 7, 20 degrees C using a dye laser with a 30-ns square-wave pulse. The results were compared to the corresponding parameters for sperm whale myoglobin and the isolated alpha and beta subunits of human hemoglobin (Olson, J.S., Rohlfs, R.J., and Gibson, Q.H. (1987) J. Biol. Chem., 262, 12930-12938). The rate-limiting step for O2, NO, and isonitrile binding to all five proteins is ligand migration up to the initial geminate state, and the rate of this process determines the overall bimolecular association rate constant for these ligands. In contrast, iron-ligand bond formation limits the overall bimolecular rate for CO binding. The distal pockets in leghemoglobin and in Glycera HbII are approximately 10 times more accessible kinetically to diatomic ligands than that in sperm whale myoglobin. This difference accounts for the much larger association rate constants (1-2 x 10(8) M-1 s-1) that are observed for O2 and NO binding to leghemoglobin and Glycera HbII. The rates of isonitrile migration through leghemoglobin are also very large and indicate a very fluid or open distal structure near the sixth coordination position. In contrast, there is a marked decrease in the rate of migration up to and away from the sixth coordination position in Glycera HbII with increasing ligand size. These results were also used to interpret previously published rate constants and quantum yields for the high (R) and low (T) affinity states of human hemoglobin. In contrast to the differences between the monomeric proteins, the differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.
Highlights
(1-2 X lo8 M” s-I) that are observed for O2 and NO protein is unusually low (Parkhurst et al, 1980)
Appleby (Appleby, 1984).The results listed forsperm whale myoglobin and the isolated LY and p subunits of human hemoglobin were taken from earlier published papers; in some cases the actual nanosecond laser photolysis experiments were repeated concurrently with the leghemoglobin and Glycera HbII studies in order to make direct comparison
In their measuring the rate of B A transition in highviscosity analysis of 02-rebindingdata, Stetzkowski et al (1985)assumed that solvents anda t low temperatures where the ligand is“frozen” in the distalpocket. Results obtained under these conditions are extrapolated to room temperature. Their values of kz for the CO complexes of sperm whale myoglobin and the intrinsic photochemical yield of the first geminate state is quite low, 0.1-0.2 for both leghemoglobin and myoglobin, whereas in our analysis the intrinsic photochemical yield has either been assumed or shown to be 1.0 for all ligands
Summary
Marine bloodworms,G. dibranchiata, were purchased from Woods Hole Marine Biology Laboratory. A. Appleby (Appleby, 1984).The results listed forsperm whale myoglobin and the isolated LY and p subunits of human hemoglobin were taken from earlier published papers; in some cases the actual nanosecond laser photolysis experiments were repeated concurrently with the leghemoglobin and Glycera HbII studies in order to make direct comparison. These previously (Gibson et al, 1986);the heme concentrations were in the parameters define an overall quantum yield of 0.10 which range 20-50 p ~an;d 1-mm pathlength cuvettes were used. As taken from Mims et al (1983) or remeasuredusing stopped-flowrapid shown in Fig. 2C,the absolute value of k3 affects the mixing or ordinary photolysis techniques
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