Ligand preference and orientation in b‐ and c‐type heme‐binding proteins

Abstract

Hemes are often incorporated into designed proteins. The importance of the heme ligand type and its orientation is still a matter of debate. Here, heme ligands and ligand orientation were investigated using a nonredundant (87 structures) and a redundant (1503 structures) set of structures to compare and contrast design features of natural b- and c-type heme-binding proteins. Histidine is the most common ligand. Marked differences in ligation motifs between b- and c-type hemes are higher occurrence of His-Met in c-type heme binding motifs (16.4% vs. 1.4%) and higher occurrence of exchangeable, small molecules in b-type heme binding motifs (67.6% vs. 9.9%). Histidine ligands that are part of the c-type CXXCH heme-binding motif show a distinct asymmetric distribution of orientation. They tend to point between either the heme propionates or between the NA and NB heme nitrogens. Molecular mechanics calculations show that this asymmetry is due to the bonded constraints of the covalent attachment between the heme and the protein. In contrast, the orientations of b-type hemes histidine ligands are found evenly distributed with no preference. Observed histidine heme ligand orientations show no dominating influence of electrostatic interactions between the heme propionates and the ligands. Furthermore, ligands in bis-His hemes are found more frequently perpendicular rather than parallel to each other. These correlations support energetic constraints on ligands that can be used in designing proteins.

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

“…To our knowledge, only one example of Gln-Fe coordination (for an engineered cytochrome c peroxidase [40,41]) and one example of His/Asn coordination (for a c cytochrome from Rhodobacter sphaeroides [42]) have been documented. The rarity of CONH2 coordination is confirmed in a recent survey of heme proteins [43]. Tyr22 (B10) was therefore considered for direct interaction with the ferric iron in S6803 rHb-R.…”

Prediction of Heme Binding Sites in Heme Proteins Using an Integrative Sequence Profile Coupling Evolutionary Information with Physicochemical Properties

“…The training set consists of 75 heme protein chains, derived from the nonredundant dataset of 89 heme proteins prepared by Fufezan et al [9]. After removing 14 chains whose HET group codes are not labeled as "HEM", we obtained the remaining 75 heme protein chains as the training set (PHeme-75).…”

“…For training and testing, we used the datasets of heme proteins in previous studies [3,9]. The training set consists of 75 heme protein chains, derived from the nonredundant dataset of 89 heme proteins prepared by Fufezan et al [9].…”

In-Cell Enzymology To Probe His–Heme Ligation in Heme Oxygenase Catalysis

“…Cys, His, Tyr, Lys, and Met) are utilized within the binding pockets of distinct classes of heme-binding proteins to coordinate the iron atom of heme, 7 all known heme oxygenases, including the non-canonical heme-degrading enzymes from S. aureus (IsdG/I) 8 and M. tuberculosis (MhuD), 9 use a strictly conserved His residue as the proximal heme ligand. 6, 10 Coordination of iron by the imidazole side-chain of histidine has been regarded as an essential interaction for HO catalysis, based on the observation that His-to-Ala mutants fail to convert heme to biliverdin in vitro despite still binding heme 11–14 and, in the case of HmuO from C. diphtheriae, catalyzing formation of the verdoheme intermediate.…”

“…13, 15, 16 Furthermore, Cys and Tyr mutants of the proximal heme ligand have also been reported to lack HO activity in vitro , 14 despite the ability of these residues to serve as heme ligands in other types of hemoproteins. 7 These observations have suggested that the His-heme interaction is unique in its ability to support HO activity and is thus required for the conversion of heme to biliverdin within the HO active site.…”