Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates their functions. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus ba3 (Tt ba3) is ~10-times faster than in the bovine enzyme, indicating inherent structural differences that affect ligand access in these enzymes. Using x-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in the Tt ba3 mutants Y133W, T231F, and Y133W&T231F, in which tyrosine and/or threonine in the O2-channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine present in the aa3 enzymes. NO binding in Y133W and Y133W&T231F is 5-times slower than in wild-type ba3 and the T231F mutant, and the results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme there is a hydrophobic “way-station,” which may further slow ligand access to the active site. Classical simulations of Xe diffusion to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W&T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

A key goal in HIV vaccine design remains to elicit broadly neutralizing antibodies (bnAbs) against the membrane-embedded envelope glycoprotein spike (mEnv). However, mEnv has lagged behind engineered soluble Envs in vaccine development due to low expression yields and the presence of extraneous proteins on particles. Here, we describe a mEnv vaccine platform that requires no extra proteins or protein engineering. MEnv trimers were fixed, purified and combined with liposomes in mild detergent. On removal of detergent, mEnvs were observed embedded in particles, designated mEnv liposomes (MELs), which were recognized by HIV bnAbs but not non-nAbs. Following sequential immunization in rabbits, MEL antisera neutralized select tier 2 HIV isolates. Variations between the Env immunogens, including a missing N-glycosylation site at position 197 near the CD4 binding site, provide insights into the specificities elicited and possible ways to improve immunogens. MELs can facilitate vaccine design to elicit HIV bnAbs using biochemically defined and multimerized mEnv.