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.cytochrome c oxidase | oxidative phosphorylation | resonance Raman spectroscopy | ATP | oxygen

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a 3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.bioenergetics | X-ray free electron laser | crystallography | cytochrome c oxidase | serial femtosecond crystallography

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain. It reduces oxygen to water and harnesses the released energy to translocate protons across the inner mitochondrial membrane. The mechanism by which the oxygen chemistry is coupled to proton translocation is not yet resolved owing to the difficulty of monitoring dynamic proton transfer events. Here we summarize several postulated mechanisms for proton translocation, which have been supported by a variety of vibrational spectroscopic studies. We recently proposed a proton translocation model involving proton accessibility to the regions near the propionate groups of the heme a and heme a3 redox centers of the enzyme based by hydrogen/deuterium (H/D) exchange Raman scattering studies (Egawa et al., PLOS ONE 2013). To advance our understanding of this model and to refine the proton accessibility to the hemes, the H/D exchange dependence of the heme propionate group vibrational modes on temperature and pH was measured. The H/D exchange detected at the propionate groups of heme a3 takes place within a few seconds under all conditions. In contrast, that detected at the heme a propionates occurs in the oxidized but not the reduced enzyme and the H/D exchange is pH-dependent with a pKa of ~8.0 (faster at high pH). Analysis of the thermodynamic parameters revealed that, as the pH is varied, entropy/enthalpy compensation held the free energy of activation in a narrow range. The redox dependence of the possible proton pathways to the heme groups is discussed.

Cysteine desulfurase enzymes catalyze sulfur mobilization from l‐cysteine to sulfur‐containing biomolecules such as iron‐sulfur (Fe‐S) clusters and thio‐tRNAs. The enzymes utilize the cofactor pyridoxal‐5'‐phosphate (PLP), which forms the external substrate‐ and product‐aldimines and ketimines during catalysis and are grouped into two types (I and II) based on their different catalytic loops. To clarify the structure‐based catalytic mechanisms for each group, we determined the structures of the external substrate‐ and product‐aldimines as catalytic intermediates of NifS (type I) and SufS (type II) that are involved in Fe‐S cluster biosynthesis using X‐ray crystallographic snapshot analysis. As a common intermediate structure, the thiol group of the PLP‐l‐cysteine external aldimine is stabilized by the conserved histidine adjacent to PLP through a polar interaction. This interaction makes the thiol group orientated for subsequent nucleophilic attack by a conserved cysteine residue on the catalytic loop in the state of PLP‐l‐cysteine ketimine, which is formed from the PLP‐l‐cysteine aldimine. Unlike the intermediates, structural changes of the loops were different between the type I and II enzymes. In the type I enzyme, conformational and topological change of the loop is necessary for nucleophilic attack by the cysteine. In contrast, the loop in type II cysteine desulfurase enzymes showed no large conformational change; rather, it might possibly orient the thiol group of the catalytic cysteine for nucleophilic attack toward PLP‐l‐cysteine. The present structures allow a revision of the catalytic mechanism and may provide a clue for consideration of enzyme function, structural diversity, and evolution of cysteine desulfurase enzymes. Database Structural data are available in PDB database under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5WT2, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5WT4, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZSP, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZST, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZS9, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZSK, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZSO, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6KFZ, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6KG0, and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6KG1.

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.

In efforts to probe the function of the O2-reduction site of bovine heart resting oxidized cytochrome c oxidase, highly sensitive resonance Raman analyses of azide binding to the O2-reduction site show that azide is simultaneously accessible to Fe and Cu ions in the O2-reduction site. This indicates that the site has spatial multiplicity for accepting significantly larger external ligands than expected from its X-ray structure. This could contribute to the accurate coupling between the O2 reduction and the proton pump.

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.