Cytochrome c oxidase (CcO), a heme-containing enzyme in the mitochondrial respiratory chain, catalyzes the reduction of molecular oxygen (O2) to water while pumping protons across the inner membrane to drive ATP synthesis. Under hypoxic conditions, sustaining ATP production is critical for cell survival. A recent study identified Higd1A, a mitochondrially encoded 10.4 kDa membrane protein, as a positive regulator of CcO that enhances ATP levels under hypoxic stress.1 However, its regulatory mechanism remains unclear. To investigate how Higd1A enhances CcO activity, we reconstituted CcO-Higd1A complexes into proteoliposomes, and measured proton pumping and O₂ reduction rates of CcO in the presence of Higd1A. Proton pump activity, assessed by pH changes using phenol red, increased by a factor of 2.5 due to Higd1A. In contrast, O2 reduction, monitored via O2 consumption using hemoglobin, increased only 1.2-fold. These findings suggest that Higd1A preferentially enhances proton pumping rather than electron transfer efficiency, leading to an increase in proton-to-electron stoichiometry. Previous resonance Raman spectroscopy analysis revealed that Higd1A induces a structural change near heme a, a key component of the putative proton-pump H-pathway in CcO.1 This structural alteration likely optimizes proton transfer, enhancing ATP production under hypoxia and helping cells adapt to oxygen-limiting conditions.