Second coordination sphere influences on heme electronic structure and reactivity in hemoproteins

Abstract A large variety of oxygen-binding hemoproteins with quite different functions share the common iron protoporphyrin IX prosthetic group axially ligated by a histidyl imidazole. To the degree that the members of this class of proteins differ in reactivities and electronic/magnetic properties, they must therefore reflect wholly influences of the second coordination sphere, i.e. the highly folded polypeptide chain. These influences can exert themselves by varying the axial imidazole bonding or by modulating the porphyrin π electronic cloud via Van der Waals interactions. Perhaps the most subtle protein effect is to differentiate between the two isomers which result from axial coordination of the imidazole or the two sides of the heme (Fig. 1). Changing the location of the axial imidazole interchanges the position of methyl-1 with vinyl-4 and vinyl-2 with methyl-3. While all X-ray crystallographic analyses have proposed a single heme orientation for each protein studied to date [1–3], proton NMR evidence [4–8] from our laboratory has demonstrated that a large number of b-type hemoproteins, in fact, exist in solution as a mixture of two species, differing by the rotation of the heme about the α-γ-meso axis. This heme orientational or rotational disorder has been characterized for the monomeric Chironomus hemoglobin [5], cytochrome b 5 [7], both native and reconstituted myoglobin [4, 8], and reconstituted horseradish peroxidase [6]. In other cases, heme rotational disorder can be induced by reconstitution of the protein [9]. Thus the initial reaction between apo-myoglobin and hemin yields a 1:1 mixture of the two heme orientations depicted in Fig. 1, and equilibration to the native protein is very long (⩾30 min) at physiological Ph. It is therefore important to assess the influence of the heme orientation (or the altered second coordination sphere) on the properties of the individual proteins. We have studied two properties of such disordered proteins: the position of the high-spin, low-spin equilibrium in the met-azide forms of native disordered Chironomus hemoglobins, and the oxygen affinity of the kinetically trapped disordered sperm whale myoglobin. Using the average heme methyl hyperfine shift as an index of the position of the high-spin, low-spin equilibrium [10], we find that the Chironomus met HbN 3 exhibits as significantly larger high-spin contribution for the heme orientation in A of Fig. 1, which dictates that in this orientation the iron experiences a weaker axial ligand field than in orientation B. In sperm whale metMbN 3 , heme orientation A also yields a weaker axial ligand field, but by a much reduced margin. Qualitative estimates of the difference in oxygen affinity for the two heme orientations for a given polypeptide chain have been made for sperm whale myoglobin. Reduction of the 1:1 mixture of heme orientations resulting from an in situ reconstitution yields an average oxygen half-saturation pressure a factor of 3 smaller than for the native protein or the 1:1 mixture after equilibration of the heme orientation [11]. Thus the protein with the ‘wrong’ orientation appears to have an oxygen affinity greater by an order of magnitude than for the X-ray determined orientation [2]. Preliminary evidence indicates that myoglobin with the ‘wrong’ heme orientation may, in fact, be physiologically functional.