CHAPTER 7

DISCUSSION

When describing the conformation of myoglobin, the overall structure as defined by the average location of the constituent atoms should be considered. As a consequence of increasing temperature, progressive changes in the structural and functional properties of myoglobin have been monitored electrochemically. Usually the amplitudes of atomic motion vary while the average atomic positions stay constant. However, an abrupt change in the temperature dependence of the structural and functional parameters was monitored resulting from a new set of average atom positions.¹

From 5-37° C, the temperature dependence of the formal potential of the heterogeneous electron transfer reaction of myoglobin can be attributed to discrete structural fluctuations which occur within the protein. At 37° C, the non-spontaneous disruption of the heme is followed by the spontaneous complete unfolding and denaturation of the protein. Experimentally, the loss of the heme and the denaturation of the protein are indicated by the positive reaction center entropy trend from 40-50° C.

The results for the temperature dependence of the rate constant for the electron transfer reaction of myoglobin on the basis of these experiments are not conclusive. The separation in peak potentials is an indication of the reversibility of a reaction. These experiments are indicative of quasi-reversible to irreversible behavior. In the calculation of the heterogeneous rate constant, using the Nicholson plot,² the kinetic parameter, y , cannot be adequately determined on the basis of the peak separation potentials reported here. Conformational transitions and the hydrophobicity of the electrode-solution interface will directly impact the rate of electron transfer; however, the exact nature of these effects remains to be elucidated. Single potential step chronoabsorptometry (SPS/CA) would be an alternate method to further investigate the temperature dependence of the rate constant of the heterogeneous electron transfer reaction of myoglobin.³

Pathways for electron tunneling in biological systems have been modeled on the basis of protein-mediated electronic coupling calculations.⁴ A physical tunneling pathway is a collection of interacting bonds in a protein around and between the donor and acceptor that make some contribution to the donor-acceptor interaction. Myoglobin is a highly helical protein. The best family of pathways is between HIS 81 and the porphyrin are shown in Figure 65. The paths follow the a -helix from the HIS to the porphyrin and the though-space connections onto the ring. In myoglobin, a good number of pathways exist which differ from each other in minor ways. Therefore, changing specific protein atoms or bonds will induce a change in the rate of heterogeneous electron transfer.

The relatively large number of electron tunneling paths characteristic of myoglobin is not found in other proteins. For example, in cytochrome c, only about 10-30 strongly coupled pathways are found, most without any through-space connections. The limited electron tunneling pathways in cytochome c can be attributed to the less helical and less compact structure. However, the measured rates in cytochrome c are known with greater certainty because they are sufficiently fast. Furthermore, these effective transfer distances track quite well with the measured rates.

The experiments presented here should be applied to hemoglobin, another important oxygen transporting protein. Found in red blood cells, hemoglobin serves as the oxygen carrier in blood and plays a vital role in the transport of carbon dioxide and hydrogen ion. The protein consists of four polypeptide chains held together by noncovalent interactions. Each polypeptide chain contains a heme group and a single oxygen-binding site. Hemoglobin A, the predominant hemoglobin in adults, consists of two alpha (a ) chains and two beta (b ) chains. Interestingly enough, the b chain of hemoglobin and the main chain of myoglobin are strikingly similar (Figure 66).⁵Although the amino acids are identical at only 24 of 141 positions, the three-dimensional folding of their main chains reveals a close resemblance.  

1. Yuan, X.; Hawkridge, F.M.; Chlebowski, J.F. J. Electroanal. Chem. 1993, 350, 29-42.

2. Nicholson, R.S. Anal. Chem. 1965, 37, 1351-1355.

3. King, B.C.; Hawkridge, F.M. J. Electroanal. Chem. 1987, 237, 81-92.

4. Electron Transfer in Inorganic, Organic, and Biological Systems. (J.R. Bolton, N. Mataga; G. McLendon, Eds.) "Developed from a symposium sponsored by the International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii, December 17-22, 1989."

5. Stryer, L. Biochemistry. New York: W.H. Freeman and Company, 1988.