Paramagnetic ¹³C and ¹³N NMR analyses of cyanide- (¹³C¹³N-) ligated ferric peroxidases: The push effect, not pull effect, modulates the compound formation rate

Paramagnetic ¹³C and ¹⁵N NMR spectroscopy of heme-bound cyanide (¹³C¹⁵N) was utilized to quantitatively distinguish the electron donor effect (the push effect) from the proximal histidine and hydrogen-bonding effect (the pull effect) from the distal amino acid residues in cytochrome c peroxidase (CcP), ascorbate peroxidase (APX), lignin peroxidase (LiP), and manganese peroxidase (MnP). Paramagnetic ¹³C NMR signals of heme-bound ¹³C¹⁵N of these peroxidases were observed in a wide range, -3501 ppm (CcP), -3563 ppm (APX), -3823 ppm (MnP), and -3826 ppm (LiP), while paramagnetic ¹⁵N NMR signals of those were detected in a narrow range, 574 ppm (ARP), 605 ppm (CcP), 626 ppm (LiP), and 654 ppm (MnP). Detailed analysis, combined with the previous results for horseradish peroxidase and Arthromyces ramosus peroxidase, indicated that the push effect is quite different among these peroxidases while the pull effect is similar. More importantly, a strong correlation between the ¹³C NMR shift (the push effect) and the compound I formation rate was observed, indicating that the push effect causes a variation in the compound I formation rate. Comparison of the ¹³C and ¹⁵N NMR results of these peroxidases with their crystal structures suggests that the orientation of the proximal imidazole plane to the heme N - Fe - N axis controls the push effect and the compound I formation rate of peroxidase.