The Association Rate Constant for Heme Binding to Globin Is Independent of Protein Structure

TLDR

The study measured CO‑heme association rates across 35 recombinant apomyoglobins and other apoproteins to elucidate factors influencing heme affinity and binding velocity. Binding proceeds via initial nonspecific heme–protein complexes that dissociate, followed by specific pocket binding where the heme pocket collapses and forms a bond with proximal His93. The CO‑heme association rate is universally ~1 × 10⁸ M⁻¹ s⁻¹ across all proteins, while variations in overall affinity (≈1 × 10¹⁴ M⁻¹ for sperm whale apomyoglobin) arise mainly from dissociation kinetics and contributions of nonspecific hydrophobic, covalent, and specific pocket interactions.

Abstract

Rate constants for CO-heme binding to 35 different recombinant apomyoglobins and several other apoproteins were measured in an effort to understand the factors governing heme affinity and the velocity of the association reaction. Surprisingly, the rate constant for the binding of monomeric heme is ≈1 × 108 M-1 s-1 regardless of the structure or overall affinity of the apoprotein for iron−porphyrin. Major differences between the proteins are reflected primarily in the rates of dissociation of the prosthetic group. Slow phases observed in the reaction of CO heme with excess apomyoglobin result from formation of nonspecific heme−protein complexes which must dissociate before heme can bind specifically in the heme pocket. Once the specific heme−globin complex is formed, the heme pocket rapidly collapses around the porphyrin, simultaneously forming the bond between the proximal His93 and the heme iron atom. The overall affinity of sperm whale apomyoglobin for hemin is ∼1 × 1014 M-1. Nonspecific hydrophobic interactions between the porphyrin and the apolar heme cavity account for a factor of 105−107. Covalent bond formation between Fe3+ and His93(F8) provides an additional factor of 103−104. Specific interactions with conserved amino acids in the heme pocket contribute the final factor of 103−104.