Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.

Researchers studied the kinetics of reconstitution for horse heart myoglobin with synthetic hemins. They determined that the orientation of heme plays a significant role in the process and identified correlations between the shape and intensity of dichroic bands and heme orientation.

Research Objective

• The primary aim of this research was to further our understanding of the reconstitution kinetics of horse heart myoglobin with synthetic hemins. Specifically, the research aimed to provide new insights into the heme re-orientation process following the heme insertion into the globin pocket.

Methodology

• The researchers used circular dichroism to investigate the reconstitution kinetics of horse heart myoglobin with two synthetic hemins, in which the 6- or the 7-propionate is replaced by a methyl group.
• The focus was on the heme orientation inside the reconstituted protein.

Key Findings

• The study confirmed that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, underscoring the importance of the salt bridge between this propionate and the basic CD3 residue of the apoprotein.
• It was found that there are correlations between the shape and intensity of the dichroic bands and the heme orientation inside the reconstituted protein.
• Positive Soret CD bands correlated with hemoglobins that possess the heme in the ‘correct’ orientation, while negative dichroic bands are associated with hemoglobins that possess the heme in the ‘reversed’ orientation.
• The negligible contribution to the CD signal in reconstituted proteins can be associated to a ‘wrong’ orientation of the heme group, regardless of whether the orientation is ‘correct’ or ‘reversed’.
• The results suggested that the disturbances due to the heme re-orientation appear as a local phenomenon, not influencing the distant domains of the macromolecule.

Significance of the Findings

• These findings enhance our understanding of the heme re-orientation process after the insertion into the globin pocket, which can contribute to further studies in related biochemistry fields.
• The revealed correlation between the shape and intensity of the dichroic bands and the heme orientation might provide a novel perspective for protein research.
• Moreover, it highlights the importance of correct heme orientation for its functional performance in hemoglobins and may enrich studies on protein structure and function.