The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin

Abstract

The grail of protein science is the connection between structure and function. For myoglobin (Mb) this goal is close. Described as only a passive dioxygen storage protein in texts, we argue here that Mb is actually an allosteric enzyme that can catalyze reactions among small molecules. Studies of the structural, spectroscopic, and kinetic properties of Mb lead to a model that relates structure, energy landscape, dynamics, and function. Mb functions as a miniature chemical reactor, concentrating and orienting diatomic molecules such as NO, CO, O(2), and H(2)O(2) in highly conserved internal cavities. Reactions can be controlled because Mb exists in distinct taxonomic substates with different catalytic properties and connectivities of internal cavities.

Figure 1

(a) The sperm whale Mb skeleton, with side chains Tyr-103, -146, and -151, Trp-7 and -14, Met-55 and -131, His-64 and -93, and Xe1, Xe2, Xe3, and Xe4 from ref. . (b) Cartoon of Mb as NO is reacting with a bound O₂. The heme cavity is on the distal (Upper), the Xe1 cavity on the proximal (Lower) side of the heme. A dioxygen molecule is shown bound covalently to the iron atom in the heme cavity. A NO molecule can move from the Xe1 cavity via the path shown to react with the O₂.

Figure 2

X-ray-determined structure of left Mb at pH 5 (Protein DataBank file 1spe, from ref. 38), and right Mb at pH 7 (Protein Data Bank file 1a6g, from Ref. 23). His-64 (Upper Left) has moved outside the heme pocket in A₀, and smaller changes are visible throughout the protein. The B-site is labeled B, and the xenon cavities 1, 2, 3, and 4 are labeled with numbers.

Figure 3

(a) Tree diagram of the taxonomic substate of Mb and its numerous statistical substates. (b) Cross section through the hierarchical energy landscape. (c) Infrared spectrum of CO bound to the iron atom of Mb, showing the stretch frequencies of the three A substates. Allostery is a consequence of the existence of these taxonomic substates.

Figure 4

Kinetics of MbO₂ oxidation by nitrite. (a) Kinetics of oxidation of 20 μM MbO₂, observed when MbO₂ is mixed with 0.1% NO (wt/vol) in a 50 mM sodium acetate/HCl buffer ≈5 min after exposure of deoxyMb to air to produce MbO₂. Complete UV-Vis optical absorbance spectra were obtained on a dual-beam OLIS (Jefferson, GA) stopped-flow apparatus. Filling of the chamber required 20 ms. (b) pH dependence of these kinetics.