Influence of cysteine-directed mutations at the Ω-loops on peroxidase activity of human cytochrome c

Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions with Neuroglobin

“…The structural perturbations by I81N substitution may affect the inherent peroxidase activity of Cyt c because a tight and stable conformation is essential to keep it at a low peroxidase activity level in physiological conditions . As recently reported, Cyt c samples the high-spin conformers more frequently under acidic conditions . Meanwhile, the intermembrane space of mitochondria is an acidic pH environment (6.88 ± 0.09) and the intracellular pH decreases during apoptosis. ,, Thus, it is physiologically relevant to investigate the relationship between the peroxidase activity of I81N h Cyt c and the acidic pH change.…”

“… 28 As recently reported, Cyt c samples the high-spin conformers more frequently under acidic conditions. 43 Meanwhile, the intermembrane space of mitochondria is an acidic pH environment (6.88 ± 0.09) and the intracellular pH decreases during apoptosis. 10 , 38 , 44 Thus, it is physiologically relevant to investigate the relationship between the peroxidase activity of I81N h Cyt c and the acidic pH change.…”

Structural Dynamics of the Precatalytic State of Human Cytochrome c upon T28C, G34C, and A50C Mutations: A Molecular Dynamics Simulation Perspective

“…Plots showing correlation of catalytic efficiency to distances between heme iron and the center of mass of corresponding Ω-loops (upper panels (from left to right): Fe–(12–28 Ω-loop), Fe–(29–39 Ω-loop), Fe–(40–57 Ω-loop), and Fe–(71–85 Ω-loop)) and Cavity A (lower panel, left), Cavity B (lower panel, middle), and Fe–Arg38 (lower panel, right). Data of the WT and P76C were previously reported. , The peroxidase activity was performed by ABTS assay when 2,2-azinobis(3-ethylbenthiazoline-6-sulfonic acid), ABTS, and H 2 O 2 were used as substrates. Catalytic efficiency was calculated as bold-italick boldc bolda boldt boldA boldB boldT boldS / bold-italicK bold-italicM boldA boldB boldT boldS , where bold-italick boldc bolda boldt boldA boldB boldT boldS and bold-italicK bold-italicM boldA boldB boldT boldS are the Michalis–Menten parameters where the concentration of ABTS is varied in the ABTS assay.…”

“…Specifically, the ESR signals from the spin-labeled proteins allow us to directly determine the rotational correlation time, τ R , at the labeled sites. , This conventional ESR approach is sensitive to the local flexibility due to molecular rotational motion and is suited for small compact proteins . Our previous experimental data , showed that the flexibility at the site of mutations obtained from the ESR results was found in the following order: P76C > G34C > A50C > T28C hCtyc. This trend was consistent with the peroxidase activity of the proteins, where the activity of the WT was comparable to that of the T28C mutant (Table S1).…”

“…We have examined the cysteine-modified mutated hCytc at four random positions: T28C and G34C in the proximal Ω-loop, A50C in the central Ω-loop, and P76C in the distal Ω-loop. 20 , 21 The advantage of preparing the cysteine side chain was the capability to examine the dynamics at the mutated site by spin labeling (SL) combined with electron spin resonance (ESR). Specifically, the ESR signals from the spin-labeled proteins allow us to directly determine the rotational correlation time, τ R , at the labeled sites.…”