Masahide Hikita

Higd1a is a positive regulator of cytochrome c oxidase.

Significance We identified hypoxia-inducible domain family, member 1A (Higd1a) as a positive regulator of cytochrome c oxidase (CcO). CcO, the terminal component of the mitochondrial electron transfer system, reductively converts molecular oxygen to water coupled to pump protons across the inner mitochondrial membrane. Higd1a is transiently induced under hypoxic conditions and increases CcO activity by directly interacting with CcO in the vicinity of its active center. Induction of Higd1a leads to increased oxygen consumption and subsequent mitochondrial ATP synthesis, thereby improving cell viability under hypoxia. 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.

Resonance Raman Spectral Properties of FMN of Bovine Heart NADH:ubiquinone Oxidoreductase Suggesting a Mechanism for the Prevention of Spontaneous Production of Reactive Oxygen Species

A highly improved method for obtaining resonance Raman (RR) spectra provided spectra comparable to the best known flavoprotein spectra when the method was tested using bovine heart NADH:ubiquinone oxidoreductase (Complex I), a protein with a molecular mass of 1000 kDa, which causes the level of RR noise to be 1 order of magnitude higher than for most flavoproteins. The FMN RR band shift (1631/1633 cm(-1)) and the increase in the magnitude of the band at 1252 cm(-1) upon binding to Complex I suggest hydrogen bond formation involving one of the C=O groups [C(2)=O] of isoalloxazine to stabilize its quinoid form. This lowers the redox potential of FMN and the electron density of the O(2) binding site [a carbon atom, C(4a)] in the reduced form. Thus, spontaneous production of reactive oxygen species at the FMN site is prevented by minimizing the duration of the fully reduced state by accelerating the FMN oxidation and by weakening the O(2) affinity of C(4a). Other band shifts (1258/1252 cm(-1) and 1161/1158 cm(-1)) suggest a significantly weaker hydrogen bond to the NH group [N(3)-H] of isoalloxazine. This result, together with the reported X-ray structure in which N(3)-H is surrounded by negatively charged surface without hydrogen bond formation, suggests that N(3)-H is weakly but significantly polarized. The polarized N(3)-H, adjacent to the C(2)=O group, stabilizes the polarized state of C(2)=O to strengthen the hydrogen bond to C(2)=O. This could fine-tune the hydrogen bond strength. Other results show a high-dielectric constant environment and weak hydrogen bonds to the isoalloxazine, suggesting adaptability for various functional controls.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes.