2.1 Introduction

Myoglobin, an extremely compact heme protein (MW ~ 17 800), found primarily in cardiac and red skeletal muscles, functions in the storage of oxygen and facilitates the transport of oxygen to the mitochondria for oxidative phosphorylation.1 Myoglobin is particularly abundant in diving mammals including the whale, seal, and porpoise, whose muscles are so rich, they are brown. These mammals are able to remain submerged for long periods due to the storage of oxygen by muscle myoglobin.2

A reservoir of oxygen located in the slow phasic muscle fibers, myoglobin consists of a single polypeptide chain of about 153 amino acids.3, 4, 5 The molecule contains approximately 1200 atoms (excluding hydrogen). Approximately 70% of the main chain is folded into eight major, right-handed a -helices (identified as segments A-H with the first residue of segment A being A1, etc.) The majority of the rest of the chain forms turns between helices devoid of symmetry. Four of the helices are terminated with a proline residue, whose five-membered ring does not fit within a straight stretch of the a -helix; thereby disrupting the helix6 (Figure1).

Model of Myoglobin at High Resolution

The main chain peptide groups are rigid planar units, and the carbonyl group of each is trans to the NH; therefore, all peptide bonds are in planar-trans conformation (Figure 2). The peptide group is planar because the carbon-nitrogen bond has partial double-bond character. As a result, rotation about the C-N bond is restricted.

The inside and outside surfaces of the protein are well defined.9 The interior consists almost entirely of nonpolar residues including leucine, valine, methionine, and phenylalanine (Figures 3, 4, and 5). Polar residues such as aspartate, glutamate, lysine, and arginine are absent from the interior protein surface. In fact, two histidines are the only polar residues which play an integral role in the binding of heme oxygen. The outside of the protein has both polar and nonpolar residues.

The distribution of side chains uncovers a revealing phenomenon in protein architecture.11 Hydrophobic attractions are the driving force behind protein folding. Furthermore, non-polar solute molecules associate so readily because of the fundamental properties of water. Water is highly cohesive, and hydrophobic groups are thermodynamically more stable. In other words, water bonds strongly to itself, and the water will reorient to form the maximum number of hydrogen bonds when in the presence of non-polar residues.

2.2 X-Ray Analysis

Myoglobin is hailed to be the first protein to be seen at atomic resolution. Breakthrough X-Ray analysis of myoglobin was carried out by John Kendrew and his colleagues in England in the 1950s.1 A very complex X-Ray diffraction pattern with nearly 25,000 reflections was resolved via a fourier transform computer analysis in three stages (Figure 6). During Stage I completed in 1957, John Kendrew elucidated the three-dimensional structure of myoglobin to 0.6 nm resolution.12 The model derived from their Fourier synthesis contained a set of high-density rods of just the dimensions expected for a polypeptide chain. The molecule appeared very compact (Figure 7) . The resolution of Stage II which was carried out 0.2 nm was high enough to identify most amino acid residues in the polypeptide chain.13 Closer examination showed that it consisted of a complicated and intertwining sets of these rods, going straight for a distance, then turning a corner and going off in a new direction. In Stage III, the 0.14 nm resolution uncovered the complete amino acid sequence which was also confirmed by chemical analysis.14 The location of the iron atom of the heme was also evident because it contains many more electrons than does any other atom in the structure (Figure 8).

2.3 The Heme

The capacity of myoglobin to bind oxygen depends on the presence of a heme. A heme is a nonpolypeptide prosthetic group consisting of protoporphyrin and a central iron atom. In solution, this heme group gives myoglobin its distinctive crimson hue. Proteins like myoglobin require tightly bound, specific nonpolypeptide units, termed prosthetic groups, for their biological activities (Figure 9). In apomyoglobin, the heme group is not present in the structure.

In myoglobin, the heme consists of an organic part and an iron part. The organic part, protoporphyrin, is made up of four pyrrole rings linked by methene bridges to form a tetrapyrrole ring. The iron atom in the heme binds to four nitrogen atoms in the center of the protoporphyrin ring (Figure 10). The iron can form two additional bonds on either side of the heme plane. These bonding positions are termed the fifth and sixth coordination positions (Figure 11).

The iron atom can be in the ferrous (+2) or the ferric (+3) oxidation state.

Fe(III)Mb + e- Û Fe(II)Mb

Only the Fe +2 or ferrous state can bind oxygen.

The heme group is located in a crevice in the myoglobin molecule. The highly polar propionate side chains of the heme are on the surface of the molecule. At physiological pH, these carboxylic acid groups are ionized. The rest of the heme is inside the molecule, where it is surrounded by nonpolar residues except for two histidines. The iron atom of the heme is directly bonded to the proximal histidine residue F8 which occupies the fifth coordination position. The iron atom is about 0.3 A out of the plane of the porphyrin, on the same side as histidine F8. The oxygen-binding site is on the other side of the heme plane, at the sixth coordination position. A second histidine residue (E7), termed the distal histidine, is near the heme but not bonded to it (Figure 12).

2.4 Intrinsic Stability

The oxygen-binding site comprises only a small fraction of the volume of the myoglobin molecule. Oxygen is directly bonded only to the iron atom of the heme. The polypeptide portion of myoglobin stabilizes the oxygen-binding properties of the heme group. The rate of heme loss from reduced myoglobin is extremely slow (t1/2 ~ months at room temperature, pH 7), whereas that from oxidized myoglobin is considerably faster (t1/2 ~ 1-3 days). In water, a free ferrous heme group can bind oxygen, but it only does so for a fleeting moment. The reason is that O2 very rapidly oxidizes the ferrous heme to ferric heme, which cannot bind oxygen. A complex of O2 sandwiched between two hemes is an intermediate in this reaction. Because the apomyoglobin has an extremely high affinity for heme (~3 X 1014 M-1), the holoprotein is much more stable to denaturation than the apoprotein. The bimolecular rate of hemin binding for apomyoglobin is k’H ~ 2-8 X 107 M-1S-1.15

In myoglobin, the heme group is much less susceptible to oxidation because two myoglobin molecules cannot readily associate to form a heme-O2-heme complex.16 The formation of this sandwich is sterically blocked by the distal histidine and other residues surrounding the sixth coordination site. Thus, myoglobin has created a special microenvironment that confers distinctive properties on its prosthetic group. In general, the function of a prosthetic group is modulated by its polypeptide environment.


2.5 Conformations of Myoglobin

The conformations of the three physiologically pertinent forms of myoglobin-deoxymyoglobin, oxymyoglobin, and metmyoglobin (ferrimyoglobin)-are very similar except at the sixth coordination position17, 18

The Conformation of Myglobin

Form Oxidation State 5th 6th
deoxymyoglobin +2 His F8 empty
oxymyoglobin +2 His F8 O2
metmyoglobin +3 His F8 H2O

Methods including fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR), viscosimetric, calorimetric, and electrometric titrations have been utilized to investigate the conformations of myoglobin.19, 20, 21, 22 Under physiological conditions, autooxidation precedes denaturation (Figure 13). After loss of the heme, unfolding occurs in two stages: partial unfolding of the native apoprotein (N) to a molten globule intermediate (I) and then complete disruption of all the helical segments (U). In the structural model for myoglobin denaturation, ferrimyoglobin oxidizes and loses heme, yielding apomyoglobin in the N state. The heme group of holoMb dissociates from the protein at very low pH. The N-form of holoMb observed under neutral and mildly acidic conditions (pH 4.5 - 7.0) has ~80% a -helix content. Likewise, the N-form of apoM observed under neutral and mildly acidic conditions has ~55% a -helix content. The B, C, and E helices which make up the heme pocket then unfold to give the molten globule I state in which the A, G, and H helices are still intact. The last step is the conversion of the intermediate to the completely unflolded U state. The U-forms have a small residual a -helix content and high intrinsic viscosity under highly acidic conditions (pH <2.0) indicative of a random-coil conformation. The wide ribbons indicate helical secondary structures. The narrow lines indicate unfolded strands have much more random positions than implied. The resistance of holomyoglobin to denaturation is a function of both the intrinsic stability of the apoprotein tertiary structure and the strength of the interactions with the prosthetic group.23, 24

2.6 References

1. Kendrew, J.C.; Bodo, G.; Dintzis, H.M.; Parrish, R.G.; Wyckoff, H.W.; Phillips, D.C. Nature, 1958, 181, 622.

2. Antonini, E.; Brunori, M.; Hemoglobin and Myoglobin in their Reactions with Ligands. New York: American Elsevier Publishing Company, 1971.

3. Kendrew, J.C. Sci. Amer. 1961, 205(6), 96-111.

4. Boardman, N.K.; Partridge, S.M. Biochem. J. 1955, 59, 543-552.

5. Spackman, D.H.; Stein, W.H.; Moore, S. Anal. Chem. 1958, 30, 1190-1206.

6. Edmundson, A.B.; Hirs, C.H.W. Nature, 1961, 190, 663-665.

7. Dickerson, R.E.; Geis, I. Hemoglobin: Structure, Function, Evolution, and Pathology.

Meno Park, CA: Benjamin/Cummings, 1983.

8. Stryer, L. Biochemistry. New York: W.H. Freeman & Company, 1988.

9. Takano,T. J. Mol. Biol. 1977, 110, 537-568.

10. Dayhoff, Margaret O. Atlas of Protein Sequence and Structure. Vol. 5 Washington, D.C.: National Biomedical Research Foundation, 1972.

11. Hughson, F.M.; Wright, P.E.; Baldwin, R.L. Science, 1990, 249, 1544-1548.

12. Kendrew, J.C.; Dickerson, R.E.; Strandberg, B.E.; Hart, R.G.; Davies, D.R.; Phillips, D.C.; Shore, V.C. Nature, 1960, 185, 422-427.

13. Takano, T. J. Mol. Biol. 1977, 110, 569-584.

14. Edmundson, A.B.; Hirs, C.H.W. J. Mol. Biol. 1962, 5, 663-682.

15. Hargrove, M.S.; Krzywda, S.; Wilkinson, A.J.; Dou, Y.; Ikeda-Saito, M.; Olson, J.S. Biochemistry, 1994, 33, 11767-11775.

16. Goto, Y.; Takahashi, N.; Fink, A.L. Biochemistry, 1990, 29, 3480-3488.

17. Postnikova, G.B.; Komarov, Y.E.; Yumakova, E.M. Eur. J. Biochem. 1991, 198, 223-232.

18. Postnikova, G.B.; Yumakova, E.M. Eur. J. Biochem. 1991, 198, 241-246.

19. Lim, M.; Jackson, T.A.; Anfinnud, P.A. Science, 1995, 269, 962-966.

20. Hughson, F.M.; Barrick, D.; Baldwin, R.L. Biochemistry, 1991, 30, 4113-4118.

21. Konno, T.; Morishima, I. Biochim. Biophys. Acta. 1993, 1162, 93-98.

22. DiIorio, E.E.; Yu, W.; Calonder, C.; Winterhalter, K.H.; DeSanctis, G.; Falcioni, G.; Ascoli, F.; Giardina, B.; Brunori, M. Proc. Natl. Acad. Sci. 1993, 90, 2025-2029.

23. Henry, E.R. Biophys. J. 1993, 64, 869-885.

24. Nienhaus, G.U.; Mourant, J.R.; Chu, K.; Frauenfelder, H. Biochemistry, 1994, 33, 13413-13430.