Higd1a is a positive regulator of cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.

Keywords: ATP; cytochrome c oxidase; oxidative phosphorylation; oxygen; resonance Raman spectroscopy.

Fig. 1.

Hypoxia-inducible Higd1a directly binds to highly purified cytochrome c oxidase (hpCcO). (A) Heat map of three genes (Upper) identified as relatively rapid and transiently induced in response to hypoxia in rat neonatal cardiomyocytes, compared with genes known to be hypoxia inducible (Pfkl, Hk2, Hmox1, and Vegfa) (Lower). (B) Expression of the Higd1a protein was elevated in response to hypoxia. (C) In vitro pull-down assay with amylose resin revealed direct binding between MBP-Higd1a and the hpCcO from bovine heart. Loading controls for the hpCcO and MBP-fusion proteins are shown in immunoblots for anti-CcO subunits and CBB staining, respectively. (D) MBP-Higd1a directly integrates into hpCcO. Mixed MBP-fusion proteins and hpCcO containing 0.2% n-decyl-β-d-maltoside (DM) were resolved by blue native PAGE (BN-PAGE), followed by immunoblotting with anti-Cox4 to detect CcO and anti-Higd1a to detect Higd1a.

Fig. 2.

Higd1a regulates CcO activity through the structural change of the active center in CcO. (A) CcO activity of hpCcO and hpCcO with either recombinant MBP or recombinant MBP-Higd1a. MBP-Higd1a causes an increase in CcO activity by almost twofold. Data represent the means ± SEM of five individual experiments. **P < 0.01, compared with MBP. (B) The difference in absorption spectra between MBP-Higd1a and oxidized hpCcO. MBP-Higd1a caused spectral changes at 413 and 432 nm. Intensity changes of oxidized hpCcO spectra are plotted at 1 min (red), 5 min (brown), 10 min (dark yellow), 15 min (green), 20 min (light blue), 25 min (blue), and 30 min (purple) after adding MPB-Higd1a. (C) Resonance Raman spectra of oxidized hpCcO at 0–5 min [spectrum (a)] and oxidized hpCcO mixed with MBP-Higd1a at 0–5 min [spectrum (b)]. The Inset shows the difference of the spectra [(b) − (a)].

Fig. 3.

Higd1a positively modulates mitochondrial respiration by altering CcO activity. (A, Left) Mitochondrial fraction from rat cardiomyocytes expressing shLacZ (shLZ), shHigd1a (shHig), or both shHig and adHigd1a (adHig) were subjected to the CcO activity assay. (Right) CcO activity was measured in cardiomyocytes treated with either adLacZ (adLZ) or adHig. Data represent the means of four individual experiments. (B, Left) The maximum oxygen consumption rate (max OCR) in rat cardiomyocytes transfected with the indicated adenovirus was measured after treatment with oligomycin A and fluorocarbonyl cyanide phenylhydrazone (FCCP). Knockdown of Higd1a resulted in a significant decrease in max OCR, which was rescued by exogenously expressed Higd1a. (Right) Overexpression of Higd1a significantly increased max OCR compared with the cells with adLZ (n = 20 for each group). (C) The relative ATP production rate of cardiomyocytes treated with shLZ or shHig was measured by the MASC assay (n = 6). A numerical value of ATP production at 10 min in shLZ groups is regarded as 1.0. (D) The relative ATP production rate of cardiomyocytes treated with adLZ or adHig was measured by MASC assay (n = 5). A numerical value of ATP production at 10 min in adLZ groups is regarded as 1.0. Data represent the means ± SEM; *P < 0.05, **P < 0.01.

Fig. 4.

(A) Representative sequential YFP/CFP ratiometric images of Mit-ATeam fluorescence in cardiomyocytes expressing corresponding adenovirus during hypoxia (n = 12 for adLZ, n = 23 for shHig, n = 18 for adHig). All of the measurements were normalized to the ratio at time 0 and compared between adLZ and adHig or shHig. (Scale bar, 20 μm.) (B) Cell death of cardiomyocytes treated with shHig was significantly increased compared with the control, which was rescued by addition of adHig under hypoxic conditions for 24 h (n = 12 for each group). Data represent the means of three independent cultures, ± SEM; *P < 0.05, **P < 0.01, compared with control (adLZ or shLZ).

Fig. 5.

Higd1a acts on the H pathway. Model depicting our docking simulation (side view) and its relationship with the H pathway. The model shows the location of Higd1a (magenta) in the CcO complex (white) and its relationship to R38 of cytochrome c oxidase subunit I and the formyl group of heme a, a component of the H pathway (red arrow).