Two second-sphere amino acids, Trp66 and Asn7, are essential for Mycobacterium tuberculosis MhuD- and Staphylococcus aureus IsdG-catalyzed aerobic degradation of heme to mycobilin and staphylobilin, respectively, but it has remained unclear how these residues tune the heme substrate electronic structure to promote catalytic porphyrin oxygenation and cleavage. A combined experimental and theoretical approach has been employed to address this issue where optical and magnetic resonance spectroscopies were used to probe the active site electronic structure and quantum mechanical calculations were utilized to provide a spectroscopically-validated framework for interpretation of enzyme activity data. In cyanide-inhibited MhuD, substitution of Trp66 for a smaller amino acid decreased the rate of heme degradation, as previously observed for S. aureus IsdI, but triggered a redshift of the Soret band, in contrast to the blueshift previously reported for IsdI. In azide-inhibited IsdG, substitution of Asn7 by an alanine altered the circular dichroism intensity and magnetic circular dichroism band energies, suggesting that a strong hydrogen bond exists between the Asn7 side-chain and the iron-ligating atom of a π-donor ligand. Spectroscopically-validated density functional theory modelling of the heme electronic structures suggests that Trp66 distorts the porphyrin ligand from planarity due to steric interactions, Asn7 orients the distal ligand through a hydrogen bonding interaction, and both second sphere interactions trigger spin density delocalization onto the heme meso carbons. Ultimately, from these results a picture is emerging whereby Trp66 and Asn7 work in concert to both geometrically and electronically prepare the heme substrate for catalytic oxygenation by MhuD and IsdG.

Second-sphere tuning of enzymatic activity in noncanonical heme oxygenase

249th National Meeting (2015)

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