Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Kannt, Aimo; Pfitzner, Ute; Ruitenberg, Maarten; Hellwig, Petra; Ludwig, Bernd; Mäntele, Werner; Fendler, Klaus; Michel, Hartmut

doi:10.1074/jbc.274.53.37974

Cited by 43 publications

(39 citation statements)

References 51 publications

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“…The shift in the cytochrome aa 3 peak indicates that hemes a and a 3 are in an abnormal environment. While other yeast COX assembly mutants described thus far have had a complete absence of COX activity in conjunction with a shift in the spectral peak, there are numerous descriptions of partially active and misassembled COX enzyme in studies with Paracoccus denitrificans mutants (15,23). Our results therefore represent the first such mutants identified in yeast.…”

Section: Discussionmentioning

confidence: 56%

Mutational Analysis of the Saccharomyces cerevisiae Cytochrome c Oxidase Assembly Protein Cox11p

Banting

Glerum

2006

Eukaryot Cell

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Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa 3 peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that ⌬cox11, like ⌬sco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.

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Section: Discussionmentioning

confidence: 56%

Mutational Analysis of the Saccharomyces cerevisiae Cytochrome c Oxidase Assembly Protein Cox11p

Banting

Glerum

2006

Eukaryot Cell

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“…This has been observed to lower the heme a E m by about 260 mV in P. denitrificans oxidase (108). In MCCE with the Rb.…”

Section: Resultsmentioning

confidence: 85%

“…In contrast, the good match between the calculated and measured shift in the heme a E m when the BNC is reduced provides evidence that there is only 1H + /2e - (31,61,62,108). If the BNC reduction is electroneutral, there is insufficient change in the long-range electrostatic potential to shift the E m of a residue 13 Å away.…”

Section: Discussionmentioning

confidence: 91%

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O 2 . Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a 3 and Cu B . If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK a s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu B shifts its pK a from below 7 to above 7 and so picks up a proton when heme a 3 and Cu B are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a 3 all have pK a s above 7 in all redox states, although they have only 1.6-3.5 ∆pK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK a s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu A , heme a, heme a 3 , and Cu B shows ≈2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E m ) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E m of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E m is independent of the BNC redox state.Heme-copper oxidases are the terminal electron acceptors in anaerobic organisms. These transmembrane proteins reduce dioxygen and convert the released chemical energy into an electrochemical gradient, across the eukaryotic mitochondrial membrane or the bacterial cell membrane (1-4). Cytochrome c oxidases are the most prevalent hemecopper oxidases. In this protein 4 electrons, provided by 4 cytochromes c, are used to reduce dioxygen to water. The 4 protons needed to make water are taken from the negative cytoplasmic side of the membrane, adding to the electrochemical gradient. Four additional protons are pumped across the membrane (5). Thus, each dioxygen molecule reduced by cytochrome c oxidase is coupled to the transfer of 8 charges across the membrane.The first step of electron transfer is from cytochrome c to Cu A , a dicopper center in subunit II which extends beyond the membrane on the proton release side of the protein (Figure 1). After receiving the electron, Cu A reduces the 6-coordinate, low-spin, bis-Hi...

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“…This assumption might substantiated by the excitatory effect of iron chelators on carotid body nervous activity probably hinting to a Fenton reaction fuelled by the NADPH oxidase generated ROS (Roy et al 2004). Cytochrome a592 might posses a low midpoint potential as described in bacteria (Kannt et al 1999), inducing a short cut of electron flow within the cytochrome c oxidase between Cu A and cytochrome a3-Cu B . It was assumed that this specific property would allow the regulation of intracellular calcium levels under hypoxia (Streller et al 2002) by shifting complex IV to more negative values, and hence decreased mitochondrial membrane potential (MMP) at physiological tissue PO 2 values explaining perhaps the linear dependence of carotid body oxygen consumption on PO 2 (Acker & Lü bbers 1977).…”

Section: Carotid Body (A) Heme Protein Identificationmentioning

confidence: 97%

The oxygen sensing signal cascade under the influence of reactive oxygen species

Acker

2005

Phil. Trans. R. Soc. B

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Structural and functional integrity of organ function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of organ function. Particular reductions in oxygen availability (hypoxia) caused by respiratory or blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply by anaerobic glycolysis. Complex cellular oxygen sensing systems have evolved to tightly regulate oxygen homeostasis. In response to variations in oxygen partial pressure (PO 2 ), these systems induce adaptive and protective mechanisms to avoid or at least minimize tissue damage. These various responses might be based on a range of oxygen sensing signal cascades including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low PO 2 affinity cytochrome c oxidase (aa 3 ) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl-hydroxylase and HIF asparaginyl hydroxylase called factor-inhibiting HIF (FIH-1). Thus, specific oxygen sensing cascades involving reactive oxygen species as second messengers may by means of their different oxygen sensitivities, cell-specific and subcellular localization help to tailor various adaptive responses according to differences in tissue oxygen availability.

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Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Cited by 43 publications

References 51 publications

Mutational Analysis of the Saccharomyces cerevisiae Cytochrome c Oxidase Assembly Protein Cox11p

Mutational Analysis of the Saccharomyces cerevisiae Cytochrome c Oxidase Assembly Protein Cox11p

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

The oxygen sensing signal cascade under the influence of reactive oxygen species

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