Anion-Specific Effects on the Alkaline State of Cytochrome c

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Erik Sedlák^1,2,a, Tibor Kožár^2,b, Rastislav Varhač^1,c, Andrej Musatov^3,d, and Nataša Tomášková^1,e*

¹Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, 04154 Košice, Slovakia

²Centre for Interdisciplinary Biosciences, P. J. Šafárik University in Košice, 04154 Košice, Slovakia

³Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, 040 01 Košice, Slovakia

^* To whom correspondence should be addressed.

Received August 28, 2020; Revised November 10, 2020; Accepted November 10, 2020

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Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The pK_a value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased pK_a (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c. The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.

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KEY WORDS: alkaline isomerization, Hofmeister anions, pseudo-peroxidase activity

DOI: 10.1134/S0006297921010065

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