The importance of Asn52 in the structure-function relationship of human cytochrome c

Abstract

The function of the highly conserved residue Asn52 in human cytochrome c (H-Cyt c) is not fully understood. Herein, we show that the naturally occurring variant N52S H-Cyt c has a perturbed secondary structure, with a small fraction of high-spin species. Remarkably, it exhibits an enhanced peroxidase activity by 3-8-fold at neutral pH, as well as self-oxidation in reaction with H₂O₂. This study suggests that the H-bond network mediated by Asn52 is essential to suppress the apoptotic activity of H-Cyt c under physiological conditions.

Fig. 1. Close up view of the H-bond networks around Asn52 and the heme crevice in the ferric WT H-Cyt c (PDB code 3ZCF). Asn52 was shown as orange stick. The heme iron and heme cofactor were shown as grey ball and sticks, respectively. H-bonds were shown as yellow dash lines. A structural water molecule (w) was shown as red sphere. The heme propionates 6 and 7 were referred to as hp-6 and hp-7. The residues Gly41, Tyr48, and Ala51 linked to the disease THC4 were shown as cyan sticks.

Fig. 2. Comparison of the CD spectra in the (A) far-UV region and (B) visible region. Spectra were colored as black, blue, and red for the ferric WT, N52A, and N52S H-Cyt c, respectively.

Fig. 3. EPR spectra of ferric WT (black), N52A (blue), and N52S (red) H-Cyt c (0.25 mM), collected at 20 K, 2 mW power and 9.4 GHz.

Fig. 4. Heme degradation of H-Cyt c in reaction with H₂O₂. (A) Time-dependent UV-Vis spectra of N52S H-Cyt c. The absorptions of the Soret band (409 nm) were recorded to calculate the k_obs. (B) Linear fitting of k_obs as a function of H₂O₂ concentrations. (C) Mass spectra of N52S H-Cyt c acquired before and after incubation with H₂O₂ (0.1 mM) for 10 min, and the fragments with a maximum intensity (16+ charges) were shown for clarification.

Fig. 5. Steady-state rates of oxidation as a function of guaiacol (A), ABTS (B) and TCP (D) concentration catalyzed by WT (black), N52A (blue), and N52S (red) H-Cyt c. (C) UV-Vis spectra of TCP (0.1 mM) dehalogenation catalyzed by N52S H-Cyt c (C, 5 μM) in presence of H₂O₂ (20 mM). The experiments were carried out in triplicate and the data were fitted to the Michaelis–Menten equation.