Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from Thermus thermophilus

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 14; pp. 5259 - 5264
• Main Authors: Chang, Hsin-Yang, Choi, Sylvia K, Vakkasoglu, Ahmet Selim, Chen, Ying, Hemp, James, Fee, James A, Gennis, Robert B
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 03-04-2012; National Acad Sciences
• Subjects: Biochemistry; Biological Sciences; Catalytic Domain; Cytochrome b Group - metabolism; Cytochromes; electron transfer; Electron Transport Complex IV - metabolism; Enzymes; heme; Hydrogen Bonding; Models, Molecular; mutation; Oxidases; Oxygen; Propionates; proton pump; Proton pumps; Proton Pumps - metabolism; Protons; Pumping; Spectroscopy, Fourier Transform Infrared; Thermus; Thermus thermophilus; Thermus thermophilus - enzymology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1107345109

The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba3-type oxygen reductase from Thermus thermophilus, leading from the propionates of heme a3 to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a3, and for Glu126II (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126II, and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a3, was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a3 or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a3 is a good candidate to be the proton loading site.