Investigating the Factors Influencing Oxidative Modification of Human Cytochrome c Using Girard's Reagent T as an NMR Probe

doi:10.11938/cjmr20253164

Abstract

Abstract:

Oxidative modification of cytochrome c (Cyt c) may influence the local conformation of protein, yet the mechanism by which structural alterations of Cyt c affect its degree of oxidative modification remains unclear. In this study, Girard’s reagent T (GRT) was employed as a nuclear magnetic resonance (NMR) probe to investigate the oxidative modification levels of human Cyt c under varying environmental conditions. Experimental results demonstrated that protecting lysine residues through reductive methylation effectively reduced protein oxidation. Partial unfolding of Cyt c was found to enhance its oxidative modification, while binding Cyt c with cardiolipin significantly increased the extent of oxidation. Additionally, other factors such as protein aggregation exhibited inhibitory effects on oxidative modification.

Key words: cytochrome c, oxidative modification, Girard’s reagent T, methylation, NMR

CLC Number:

O482.53

ZHANG Guangqing, ZHAN Jianhua, XIAO Xiong, ZHU Qinjun, JIANG Bin, LIU Maili, ZHANG Xu. Investigating the Factors Influencing Oxidative Modification of Human Cytochrome c Using Girard's Reagent T as an NMR Probe[J]. Chinese Journal of Magnetic Resonance, 2026, 43(1): 37-45.

Figures/Tables 7

Fig 1

Table 1

Fig 2

Fig 3

Fig 4

Fig 5

Fig 6

References 22

[1]	STADTMAN E R, LEVINE R L. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins[J]. Amino Acids, 2003, 25(3-4): 207-218. doi: 10.1007/s00726-003-0011-2 pmid: 14661084
[2]	FEDOROVA M, KULEVA N, HOFFMANN R. Reversible and irreversible modifications of skeletal muscle proteins in a rat model of acute oxidative stress[J]. Biochim Biophys Acta Mol Basis Dis, 2009, 1792(12): 1185-1193. doi: 10.1016/j.bbadis.2009.09.011
[3]	CECARINI V, GEE J, FIORETTI E, et al. Protein oxidation and cellular homeostasis: Emphasis on metabolism[J]. Biochim Biophys Acta Mol Cell Res, 2007, 1773(2): 93-104. doi: 10.1016/j.bbamcr.2006.08.039
[4]	NGUYEN A T, DONALDSON R P. Metal-catalyzed oxidation induces carbonylation of peroxisomal proteins and loss of enzymatic activities[J]. Arch Biochem Biophys, 2005, 439(1): 25-31. doi: 10.1016/j.abb.2005.04.018 pmid: 15922287
[5]	ROUMELIOTIS S, ELEFTHERIADIS T, LIAKOPOULOS V. Is oxidative stress an issue in peritoneal dialysis[J]? Semin Dialysis, 2019, 32(5): 463-466. doi: 10.1111/sdi.v32.5
[6]	DALLE-DONNE I, ROSSI R, GIUSTARINI D, et al. Protein carbonyl groups as biomarkers of oxidative stress[J]. Clin Chim Acta, 2003, 329(1-2): 23-38. doi: 10.1016/S0009-8981(03)00003-2
[7]	ESTEVEZ M. Protein carbonyls in meat systems: A review[J]. Meat Sci, 2011, 89(3): 259-279. doi: 10.1016/j.meatsci.2011.04.025 pmid: 21621336
[8]	ZHAO B B, ZHAN J H, HU Q, et al. NMR study on the mechanism of cytochrome c methionine oxidation[J]. Chinese J Magn Reson, 2023, 40(3): 246-257.
	赵蓓蓓, 占建华, 胡琴, 等. 细胞色素c甲硫氨酸氧化机制的NMR研究[J]. 波谱学杂志, 2023, 40(3): 246-257. doi: 10.11938/cjmr20222996
[9]	YIN V, MIAN S H, KONERMANN L. Lysine carbonylation is a previously unrecognized contributor to peroxidase activation of cytochrome c by chloramine-T[J]. Chem Sci, 2019, 10(8): 2349-2359. doi: 10.1039/C8SC03624A
[10]	YANG Y, STELLA C, WANG W R, et al. Characterization of oxidative carbonylation on recombinant monoclonal antibodies[J]. Anal Chem, 2014, 86(10): 4799-4806. doi: 10.1021/ac4039866 pmid: 24731230
[11]	MIRZAEI H, REGNIER F. Identification and quantification of protein carbonylation using light and heavy isotope labeled Girard's P reagent[J]. J Chromatogr A, 2006, 1134(1-2): 122-133. pmid: 16996067
[12]	ARTEMENKO K, MI J, BERGQUIST J. Mass-spectrometry-based characterization of oxidations in proteins[J]. Free Radical Res, 2015, 49(5): 477-493. doi: 10.3109/10715762.2015.1023795
[13]	CHEA E E, DEREDGE D J, JONES L M. Insights on the conformational ensemble of Cyt c reveal a compact state during peroxidase activity[J]. Biophys J, 2020, 118(1): 128-137. doi: S0006-3495(19)30934-8 pmid: 31810655
[14]	YIN V, SHAW G S, KONERMANN L. Cytochrome c as a peroxidase: activation of the precatalytic native state by H₂O₂-induced covalent modifications[J]. J Am Chem Soc, 2017, 139(44): 15701-15709. doi: 10.1021/jacs.7b07106
[15]	ADAMS-CIOABA M A, MIN J. Structure and function of histone methylation binding proteins[J]. Biochem Cell Biol, 2009, 87(1): 93-105. doi: 10.1139/O08-129
[16]	LIU Z Y, ZHOU Y, LIU J, et al. Reductive methylation labeling, from quantitative to structural proteomics[J]. Trends Anal Chem, 2019, 118: 771-778. doi: 10.1016/j.trac.2019.07.009
[17]	MILOREY B, MALYSHKA D, SCHWEITZER-STENNER R. pH dependence of ferricytochrome c conformational transitions during binding to cardiolipin membranes: Evidence for histidine as the distal ligand at neutral pH[J]. J Phys Chem Lett, 2017, 8(9): 1993-1998. doi: 10.1021/acs.jpclett.7b00597 pmid: 28418677
[18]	TOMLINSON E J, FERGUSON S J. Loss of either of the two heme-binding cysteines from a class I c-type cytochrome has a surprisingly small effect on physicochemical properties[J]. J Biol Chem, 2000, 275(42): 32530-32534. doi: 10.1074/jbc.M004022200 pmid: 10922364
[19]	HANSKE J, TOFFEY J R, MORENZ A M, et al. Conformational properties of cardiolipin-bound cytochrome c[J]. Proc Natl Acad Sci, 2011, 109(1): 125-130. doi: 10.1073/pnas.1112312108
[20]	MILAZZO L, TOGNACCINI L, HOWES B D, et al. Unravelling the non-native low-spin state of the cytochrome c-cardiolipin complex: evidence of the formation of a his-ligated species only[J]. Biochemistry, 2017, 56(13): 1887-1898. doi: 10.1021/acs.biochem.6b01281 pmid: 28277678
[21]	ZHAN J H, HU Q, ZHU Q J, et al. Track the conformational change of unlabeled yeast cytochrome c in cell homogenate using NMR[J]. Chinese J Magn Reson, 2023, 40(1): 22-29.
	占建华, 胡琴, 朱勤俊, 等. 基于磁共振的胞浆中无标记酵母细胞色素c构象变化追踪[J]. 波谱学杂志, 2023, 40(1): 22-29. doi: 10.11938/cjmr20222985
[22]	ZHAN J, ZHANG G, CHAI X, et al. NMR reveals the conformational changes of cytochrome c upon interaction with cardiolipin[J]. Life, 2021, 11(10): 1-12. doi: 10.3390/life11101031

试剂名称	纯度/浓度	生产厂家	规格
重水	氘代率99%	Sigma Aldrich	100 mL
胰蛋白胨	AR	Oxiod	500 g
酵母提取物	AR	Oxiod	500 g
氨苄青霉素	AR	Biosharp	20 g
硫酸镁	AR	国药试剂	500 g
葡萄糖	AR	国药试剂	500 g
σ-aminolevulinic acid	AR	国药试剂	100 mg
甘油	AR	国药试剂	500 mL
氯化钙	AR	国药试剂	500 g
氯化钠	AR	国药试剂	500 g
氢氧化钠	AR	国药试剂	500 g
盐酸	AR	国药试剂	500 mL
氯化钾	AR	国药试剂	500 g
磷酸二氢钠	AR	国药试剂	500 g
磷酸氢二钠	AR	国药试剂	500 g
磷酸二氢钾	AR	国药试剂	500 g
硫酸亚铁	AR	国药试剂	500 g
EDTA-2Na	AR	国药试剂	500 g
Tris	AR	国药试剂	500 g
IPTG	AR	国药试剂	25 g
氯化锰	AR	国药试剂	500 g
氯化钴	AR	国药试剂	100 g
硫酸锌	AR	国药试剂	500 g
氯化铜	AR	国药试剂	500 g
硼酸	AR	国药试剂	500 g
磷酸氢二钾	AR	国药试剂	500 g
硫酸钠	AR	国药试剂	500 g
溶菌酶	AR	Sigma Aldrich	10 g
脱氧核糖核酸酶Ⅰ	AR	Sigma Aldrich	1 mL
盐酸胍	AR	国药试剂	500 g
心磷脂	97 %	Sigma Aldrich	100 mL
吉拉德试剂T	98 %	Sigma Aldrich	100 g
DMAB	97 %	Sigma Aldrich	25 g
甲醛水溶液	30%	国药试剂	25 mL
甘氨酸	AR	国药试剂	100 g