The X-ray Crystal Structures of Wild-type and EQ(I-286) Mutant Cytochrome c Oxidases from Rhodobacter sphaeroides

Svensson-Ek, Margareta; Abramson, Jeff; Larsson, Gisela; Törnroth, Susanna; Brzezinski, Peter; Iwata, So

doi:10.1016/s0022-2836(02)00619-8

Cited by 543 publications

(683 citation statements)

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“…Removal of the carboxyl at position 132 strongly inhibits enzyme activity (2% of wild-type RsCcO) and proton pumping (8). A chain of waters between D132 and E286 can be seen in the D pathway of high-resolution structures of aa 3 -type CcO (10)(11)(12). Computational studies and molecular dynamics simulations suggest that these waters may serve in both proton transfer and proton storage (13)(14)(15).…”

Section: Resultsmentioning

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

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Resultsmentioning

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

Proc. Natl. Acad. Sci. U.S.A.

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“…Note the water chain leading from this region to E286. The figure was prepared using VMD (Visual Molecular Dynamics)(38) from the structure reported in (39). The time course of the absorbance changes at 445nm, monitoring the reduction of heme a + heme a 3 .…”

Section: Discussionmentioning

confidence: 99%

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa₃-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

et al. 2006

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Cytochrome oxidase catalyzes the reduction of O 2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all the pumped protons enter the enzyme through the D channel, and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing the oxidase activity. N139 is one of three asparagines near the entrance of the D channel where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pK a of E286, a functionally critical residue which is located 20 Å away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pK a of E286. This finding re-enforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286 which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.During each turnover of cytochrome c oxidase, eight protons are taken up from the N-side of the membrane; four protons are used for chemistry (water formation) and four protons are pumped across the membrane(1-6). Recently, a mutation in the D-channel of the aa 3 -type oxidase from Rhodobacter sphaeroides, N139D (see Figure 1), was shown to completely eliminate proton pumping, but without reducing the oxidase activity of the enzyme (7-9). Indeed, the mutant oxidase has a turnover number that is twice that of the wild type enzyme. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA similar mutation has also been reported at the equivalent position (N131D) in the oxidase from Paracoccus denitrificans (10). The N139D mutant of theR. sphaeroides oxidase has been further characterized and shown to form the same transient intermediates as observed with the wild type oxidase (8). In this reaction, O 2 binds to the reduced heme a 3 -Cu B active site and the O-O bond is split yielding hydroxide associated with Cu B 2+ and an oxoferryl form (Fe 4+ =O 2− ) of heme a 3 . This reaction involves the transfer of one electron from nearby heme a, which leaves the active site with excess negative charge. This intermediate is called the P R state of the oxidase, and it is shortlived (τ< 100 μs). The P R state is rapidly converted to the F state upon the transfer of a proton from E286 to the active site. Hence...

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“…The simulation system was constructed by starting from the x-ray structure of CcO [Protein Data Bank entry 1M56 (33)]. The protein was surrounded by a simplified polarizable membrane, as was done in ref.…”

Section: Methodsmentioning

confidence: 99%

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase

Pisliakov

Sharma

Chu

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

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Gaining detailed understanding of the energetics of the protonpumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Despite promising mechanistic proposals, most works have not related the activation barriers of the different assumed steps to the protein structure, and there has not been a physically consistent model that reproduced the barriers needed to create a working pump. This work reevaluates the activation barriers for the primary proton transfer (PT) steps by calculations that reflect all relevant free energy contributions, including the electrostatic energies of the generated charges, the energies of water insertion, and large structural rearrangements of the donor and acceptor. The calculations have reproduced barriers that account for the directionality and sequence of events in the primary PT in CcO. It has also been found that the PT from Glu-286 (E) to the propionate of heme a3 (Prd) provides a gate for an initial back leakage from the high pH side of the membrane. Interestingly, the rotation of E that brings it closer to Prd appears to provide a way for blocking competing pathways in the primary PT. Our study elucidates and quantifies the nature of the control of the directionality in the primary PT in CcO and provides instructive insight into the role of the water molecules in biological PT, showing that ''bridges'' of several water molecules in hydrophobic regions present a problem (rather than a solution) that is minimized in the primary PT.coupled electron transfer proton transfer ͉ proton pumps ͉ dielectric effects ͉ free energy calculations ͉ pK a in nonpolar regions

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The X-ray Crystal Structures of Wild-type and EQ(I-286) Mutant Cytochrome c Oxidases from Rhodobacter sphaeroides

Cited by 543 publications

References 60 publications

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

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

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa₃-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase

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The X-ray Crystal Structures of Wild-type and EQ(I-286) Mutant Cytochrome c Oxidases from Rhodobacter sphaeroides

Cited by 543 publications

References 60 publications

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

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

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa3-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase

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Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa₃-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump