Oxygen binding and nitric oxide dioxygenase activity of cytoglobin are altered to different extents by cysteine modification.

Danlei Zhou,Aiqin Luo,James Boslett,Jay L Zweier,Craig Hemann,Xiaoping Liu

doi:10.1002/2211-5463.12230

Danlei Zhou, Aiqin Luo + Show 4 more

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https://doi.org/10.1002/2211-5463.12230

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Abstract

Cytoglobin (Cygb), like other members of the globin family, is a nitric oxide (NO) dioxygenase, metabolizing NO in an oxygen (O2)‐dependent manner. We examined the effect of modification of cysteine sulfhydryl groups of Cygb on its O2 binding and NO dioxygenase activity. The two cysteine sulfhydryls of Cygb were modified to form either an intramolecular disulfide bond (Cygb_SS), thioether bonds to N‐ethylmaleimide (NEM; Cygb_SC), or were maintained as free SH groups (Cygb_SH). It was observed that the NO dioxygenase activity of Cygb only slightly changed (~ 25%) while the P50 of O2 binding to Cygb changed over four‐fold with these modifications. Our results suggest that it is possible to separately regulate one Cygb function (such as O2 binding) without largely affecting the other Cygb functions (such as its NO dioxygenase activity).

Highlights

The Cygb monomer contains two exposed cysteine residues (Cys 38 and Cys 83) that enable Cygb to form an intramolecular disulfide bond [20,21,22]
Abbreviations Absb, the absorbance at baseline or when time t approaches to infinity; Asc, ascorbate; b5, cytochrome b5; b5R, cytochrome b5 reductase; Cygb-SC, cytoglobin with thioether bonds between a cysteine residue and N-ethylmaleimide; Cygb with free sulfhydryl group (Cygb-SH), cytoglobin with free sulfhydryl group; Cygb-SS, cytoglobin with intramolecular disulfide bond; DT, dithionite; DTT, dithiothreitol
In this study, we focus on examination of the effect of modifying the sulfhydryl groups of the cysteine residues in monomeric Cygb on the affinity of O2 binding and the rate of Cygb-mediated O2-dependent nitric oxide (NO) metabolism in the presence of cellular reductants

Summary

Introduction

The Cygb monomer contains two exposed cysteine residues (Cys 38 and Cys 83) that enable Cygb to form an intramolecular disulfide bond [20,21,22]. If the rate of Cygb reduction is not altered by modification of the sulfhydryl groups in the Cygb, the NO dioxygenase activity of Cygb may not be greatly affected even under hypoxic conditions. In this study, we focus on examination of the effect of modifying the sulfhydryl groups of the cysteine residues in monomeric Cygb on the affinity of O2 binding and the rate of Cygb-mediated O2-dependent NO metabolism in the presence of cellular reductants

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