Heme Binding by the N-Terminal Fragment 1−44 of Human Growth Hormone

Abstract

Fragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes.

Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in -thalassemic erythropoiesis

“…amino acids whose side chains are known to be favored in cytochromes). 46 We propose that in early β thalassemic erythroid precursors, characterized by high levels of ROS and heme, PRDX2 targets both ROS and heme to reduce oxidative stress. In late β-thalassemic erythropoiesis, when ROS levels are still high but heme levels are reduced, ROS might become the major target of PRDX2 ( Figure 6).…”

Antibodies Use Heme as a Cofactor to Extend Their Pathogen Elimination Activity and to Acquire New Effector Functions

“…Accordingly, it has been shown previously that heme may easily bind to various proteins such as calmodulin (38), amyloid-␤ (39,40), prone protein (41), myosin (42), apolipoproteins (10), histidine-rich glycoprotein (43), and myelin basic protein (44). Interestingly, the binding of heme to unstructured or partly structured proteins induces and stabilizes the protein structure (45)(46)(47). The CD loops that build the antigen-binding sites of Igs are particularly rich in tyrosine and tryptophan residues (33,48).…”

Non-Canonical Heme-Binding Proteins

“…[150][151][152] Limited proteolysis studies showed that digestion of hGH with pepsin leads to the selective formation of peptides 1-44 and 45-191, which are predicted to be the two physiologically relevant fragments occurring in-vivo. 153 Spolaore et al 154 reported that the fragment comprising residues 1 to 44 can bind heme. Interestingly, binding of heme is specific for the 1-44 N-terminal (Nt) fragment, as no interaction was found with peptide or with the full length protein.…”

“…Ferric heme binding has an apparent affinity of 1.48 µM and a 1:1 stoichiometry. 154 Binding of heme was characterized by the formation of a low-spin, hexacoordinated complex with a sharp absorption maximum appearing at 413 nm and a broad band around 532 nm. Reduction of bound heme produced a red-shift in the Soret band to 425 nm, and the formation of two well-defined bands at 530 nm and 560 nm.…”

“…Reduction of bound heme produced a red-shift in the Soret band to 425 nm, and the formation of two well-defined bands at 530 nm and 560 nm. 154 The spectral features are typical of bis-His ligated b-type cytochromes or of His-Fe-Met coordination. The absence of a Q-band between 600 nm and 750 nm diagnostic of Met coordination in Ntheme favors bis-His as the most probable binding mode.…”