Conformational Changes and Ligand Dissociation Kinetics following Rapid Reduction of Human Aquomethemoglobin and Horse Aquometmyoglobin by Hydrated Electrons
التفاصيل البيبلوغرافية
العنوان:
Conformational Changes and Ligand Dissociation Kinetics following Rapid Reduction of Human Aquomethemoglobin and Horse Aquometmyoglobin by Hydrated Electrons
By pulse radiolysis and fast spectrometry the reduction of methemoglobin and metmyoglobin by hydrated electrons has been followed by measuring the spectral changes in the Soret region. The rate of disappearance of the hydrated electrons has also been measured at 650 nm. In the course of the reaction of methemoglobin with hydrated electrons, three different steps could be distinguished. A first step corresponds to the reduction of the heme iron itself, and appears to be bimolecular. The reaction rate constant of this step observed at 435 nm near the isoionic point (pH 7.3) and designated k435, is 5.8 x 1010 m-1 s-1. The same value was found for the rate of disappearance of the hydrated electrons, k650. This was found in spite of the fact that not all the electrons reacting with the hemeproteins react exclusively with the heme groups. This points to the existence of an activated complex between the protein and the hydrated electron. The two reaction constants do not change upon blocking the β(93)SH groups with iodoacetamide. For metmyoglobin at the isoionic point we found that k435 = k650 = 4.3 x 1010 m-1 s-1. Calculations showed that the rate of the reaction of the hydrated electron with the hemeproteins under investigation approaches a diffusion controlled limit. A second step for methemoglobin was found to be monomolecular, with a half-time of 15 ± 2 µs. It could not be detected in metmyoglobin and it disappeared in methemoglobin upon addition of inositol hexaphosphate, indicating that this step is related to a known quaternary structural change, from the R to the T state. This conformational change appears to occur as soon as one heme group in the methemoglobin molecule becomes reduced. A third step, present both in methemoglobin and metmyoglobin, appears also to be monomolecular, with half-time of about 200 µs at pH 7.3. The reaction rate constant for this step in methemoglobin shows a marked pH dependence, suggestive of a titration curve with a pK near 8. For metmyoglobin, the suggested pK is shifted to higher pH values. We conclude that this third step corresponds to the release of heme bound water at low pH, or of OH- at high pH. Oxygen-binding experiments showed that our experimental conditions were such that the Hill parameter n and the oxygen affinity for hemoglobin, reduced by hydrated electrons, remained unaffected.
