Polar Residues in Helix VIII of Subunit I of Cytochrome <i>c</i> Oxidase Influence the Activity and the Structure of the Active Site

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Crystal structures in both oxidized and reduced forms are reported for two bacterial cytochrome c oxidase mutants that define the D and K proton paths, showing conformational change in response to reduction and the loss of strategic waters that can account for inhibition of proton transfer. In the oxidized state both mutants of the Rhodobacter sphaeroides enzyme, D132A and K362M, show overall structures similar to wild type, indicating no long-range effects of mutation. In the reduced state, the mutants show an altered conformation similar to that seen in reduced wild type, confirming this reproducible, reversible response to reduction. In the strongly inhibited D132A mutant, positions of residues and waters in the D pathway are unaffected except in the entry region close to the mutation, where a chloride ion replaces the missing carboxyl and a 2-Å shift in N207 results in loss of its associated water. In K362M, the methionine occupies the same position as the original lysine, but K362-and T359-associated waters in the wild-type structure are missing, likely accounting for the severe inhibition. Spectra of oxidized frozen crystals taken during X-ray radiation show metal center reduction, but indicate development of a strained configuration that only relaxes to a native form upon annealing. Resistance of the frozen crystal to structural change clarifies why the oxidized conformation is observable and supports the conclusion that the reduced conformation has functional significance. A mechanism is described that explains the conformational change and the incomplete response of the D-path mutant.conformational gating | membrane protein structure | proton path mutants | X-ray reduction | water chain C ytochrome c oxidase (CcO) is a major contributor to and regulator of energy conservation in eukaryotic and many prokaryotic organisms. It uses four electrons from cytochrome c and four protons from the inner side of the membrane to reduce O 2 to water (1). In each catalytic cycle, four protons are also translocated across the membrane to generate a membrane potential, but the mechanistic details of coupling between proton pumping and the electron transfer events are still not fully understood (1, 2). Our recently solved crystal structures of the fully reduced form of Rhodobacter sphaeroides (Rs) CcO reveal conformational changes that were not previously observed (3), introducing mechanistic possibilities for coupling and gating of proton transfer, the significance of which we further elucidate in this report.Bacterial forms of CcO are similar in many respects to the mammalian mitochondrial CcO and their simpler subunit composition and ease of mutation have made them useful model systems for studying the mechanism of energy conservation, assuming it to be conserved. It has become increasingly clear, however, that within the large superfamily of heme-copper oxidases some bacterial forms may have solved the proton pumping problem in different ways (4, 5), and even those most closely related to eukaryotic oxidases (the aa ...

Section: Resultssupporting

confidence: 92%

Section: S142mentioning

confidence: 99%

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

Liu

Qin

Ferguson‐Miller

2011

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“…Because H282 is always hydrogen-bonded to either a serine or threonine, the T302V mutant was examined to eliminate this hydrogen bond. The severe loss of activity and spectroscopic perturbation caused by the T302V mutation reflects previous reports of placing a hydrophobic residue in the equivalent location in several A-family heme-copper oxygen reductases (T352 in E. coli cytochrome bo 3 and in R. sphaeroides cytochrome aa 3 as well as T344 in P. denitrificans cytochrome aa 3 ) (44)(45)(46)(47).…”

Section: Previous Results From Mutating the Equivalent Of D372 In A-fsupporting

confidence: 81%

“…Although an aspartate (or aspartyl) residue near propionate-A of the active-site heme is not a conserved feature of heme-copper oxygen reductases, it is commonly found in Afamily enzymes, and thus has attracted attention. Mutagenesis experiments have been performed on the equivalent residues: D399 in the P. denitrificans aa 3 -type oxygen reductase, D407 in the R. sphaeroides aa 3 -type oxygen reductase, and D407 in the Escherichia coli bo 3 -type oxygen reductase (14,27,(43)(44)(45)(46). The D399N mutation in the P. denitrificans oxidase does not significantly change the properties of the oxidase, whereas the D399L mutant has only ∼7% activity and does not pump protons (41).…”

Section: Previous Results From Mutating the Equivalent Of D372 In A-fmentioning

confidence: 99%

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba₃fromThermus thermophilus

Chang¹,

Choi

Vakkasoglu

et al. 2012

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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 ba 3 -type oxygen reductase from Thermus thermophilus , leading from the propionates of heme a 3 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 a 3 , and for Glu126 II (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126 II , and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a 3 , 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 a 3 or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a 3 is a good candidate to be the proton loading site.

“…Replacement of the Lys I 362 residue by, e.g., a Met essentially abolishes proton transfer through the K pathway, and the activity drops to <2% of that of the wild-type CytcO (23). Proton transfer from solution to the catalytic site, via the Lys I 362 residue, requires reisomerization of the side chain to reach between the low- This article is a PNAS Direct Submission.…”

mentioning

confidence: 99%

Functional interactions between membrane-bound transporters and membranes

Öjemyr

Lee

Gennis

et al. 2010

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One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated proteinprotein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the twodimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.cytochrome aa3 | electron transfer | energy transduction | membrane protein | respiration