Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity.

David Pajuelo Reguera,Tomáš Mráček,Petr Pecina,Marek Vrbacký,Alena Pecinová,Josef Houštěk,Kristýna Čunátová

doi:10.3390/cells9020443

Abstract

Cytochrome c oxidase (COX) is regulated through tissue-, development- or environment-controlled expression of subunit isoforms. The COX4 subunit is thought to optimize respiratory chain function according to oxygen-controlled expression of its isoforms COX4i1 and COX4i2. However, biochemical mechanisms of regulation by the two variants are only partly understood. We created an HEK293-based knock-out cellular model devoid of both isoforms (COX4i1/2 KO). Subsequent knock-in of COX4i1 or COX4i2 generated cells with exclusive expression of respective isoform. Both isoforms complemented the respiratory defect of COX4i1/2 KO. The content, composition, and incorporation of COX into supercomplexes were comparable in COX4i1- and COX4i2-expressing cells. Also, COX activity, cytochrome c affinity, and respiratory rates were undistinguishable in cells expressing either isoform. Analysis of energy metabolism and the redox state in intact cells uncovered modestly increased preference for mitochondrial ATP production, consistent with the increased NADH pool oxidation and lower ROS in COX4i2-expressing cells in normoxia. Most remarkable changes were uncovered in COX oxygen kinetics. The p50 (partial pressure of oxygen at half-maximal respiration) was increased twofold in COX4i2 versus COX4i1 cells, indicating decreased oxygen affinity of the COX4i2-containing enzyme. Our finding supports the key role of the COX4i2-containing enzyme in hypoxia-sensing pathways of energy metabolism.

Highlights

Cytochrome c oxidase, the terminal enzyme of the electron transport chain, is an indispensable part of mitochondrial machinery needed for ATP production in mammalian cells (OXPHOS)
Energetic metabolism of mammalian organisms is vitally dependent on aerobic respiration, with 80–90% of inhaled oxygen being utilized by cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain
Oxygen reduction to water by cytochrome c oxidase occurs under conditions of oxygen availability varying by two orders of magnitude [36], which under extreme cases may result in incomplete COX saturation

Summary

Introduction

Cytochrome c oxidase, the terminal enzyme of the electron transport chain, is an indispensable part of mitochondrial machinery needed for ATP production in mammalian cells (OXPHOS). The crucial position in the respiratory chain pathway, a large drop of Gibbs free energy during enzyme turnover, and relatively low in vivo reserve capacity [2] predispose COX to serve as a mitochondrial OXPHOS regulator. This is reflected by the emergence of numerous regulatory subunits during the evolution of individual eukaryotic lineages [3], as well as by the discovery of numerous post-translational modifications signifying that COX has become a target of signaling pathways [4]. Subunits 6a, 7a, and 8 all exist in two variants, the L

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