БИОХИМИЯ, 2021, том 86, вып. 1, с. 73–88

УДК 577.121.7

Специфические эффекты анионов на щелочное состояние цитохрома c

© 2021 Е. Седлак 1,2, Т. Кожар 2, Р. Вархач 1, А. Мусатов 3, Н. Томашкова 1*

Department of Biochemistry, Faculty of Science, P. J. Šafárik University, 04154 Košice, Slovakia; e-mail: natasa.tomaskova@upjs.sk

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

Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, 04001 Košice, Slovakia

Поступила в редакцию 28.08.2020
После доработки 10.11.2020
Принята к публикации 10.11.2020

DOI: 10.31857/S0320972521010061

КЛЮЧЕВЫЕ СЛОВА: щелочная изомеризация, анионы Хофмейстера, псевдо-пероксидазная активность.

Аннотация

Изучено специфическое влияние анионов на структуру и термостабильность, а также на пероксидазную активность нативного (состояние III) и щелочного (состояние IV) цитохрома с (cyt c) с помощью методов УФ-абсорбционной спектроскопии, собственной флуоресценции триптофана и кругового дихроизма. Термическая и изотермическая денатурация, контролируемая, соответственно, по флуоресценции триптофана и методом кругового дихроизма, предполагают более низкую стабильность cyt c в состоянии IV по сравнению с состоянием III. Значения pKa щелочной изомеризации cyt c зависят от присутствующих солей, в соответствии с эффектом Хофмейстера на стабильность белка, т.е. космотропные анионы увеличивают, а хаотропные анионы уменьшают его значение. Пероксидазная активность cyt c в состоянии III, измеренная путём окисления гваякола, показывает четкую зависимость от положения солей в ряду Хофмейстера, в то время как cyt c в щелочном состоянии не проявляет пероксидазной активности независимо от типа анионов, присутствующих в растворе. Щелочная изомеризация cyt c в присутствии 8 М мочевины, измеренная по флуоресценции Trp59, предполагает существование высокоаффинного не природного для гемового железа лиганда даже при частично денатурированной конформации белка. Конформация cyt c в щелочном состоянии в 8 М мочевине заметно модулируется специфическим эффектом анионов. Основываясь на снижении флуоресценции Trp59 при титровании до щелочного значения рН в 8 М мочевине и используя метод молекулярной динамики, мы предполагаем, что Lys79-конформер является, скорее всего, доминирующим щелочным конформером cyt c. Высокое сродство шестого лиганда к гемовому железу, вероятно, является причиной отсутствия пероксидазной активности у cyt c в щелочной среде.

Сноски

* Адресат для корреспонденции.

Финансирование

Эта работа была поддержана исследовательским грантом, предоставленным Словацким агентством исследований и разработок (грант № APVV-15-0069) и грантовым агентством Министерства образования, науки, исследований и спорта Словацкой Республики (грант № VEGA 2/0009/17). Данная публикация является результатом реализации проекта: Открытое научное сообщество для современных междисциплинарных исследований в медицине (Акроним: OPENMED), ITMS2014 +: 313011V455 при поддержке Интегрированной инфраструктуры операционной программы, финансируемой ЕФРР.

Конфликт интересов

Авторы заявляют об отсутствии прямого или потенциального конфликта интересов, связанного с публикацией этой статьи.

Соблюдение этических норм

Эта статья не содержит описания исследований, выполненных авторами с участием человека или животных в качестве объектов.

Дополнительные материалы

Приложение к статье на английском языке опубликовано на сайте журнала «Biochemistry» (Moscow) (http://protein.bio.msu.ru/biokhimiya/) и на сайте издательства Springer (https://link.springer.com/journal/10541), том 86, вып. 1, 2021.

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