Overexpression and Purification of CytochromecOxidase fromRhodobacter sphaeroides

Zhen, Yuejun; Qian, Jie; Follmann, Kara; Hayward, T.; Nilsson, Thomas; Dahn, Michael S.; Hilmi, Yasmin; Hamer, Alicia G.; Hosler, Jonathan P.; Ferguson‐Miller, Shelagh

doi:10.1006/prep.1998.0903

Cited by 71 publications

(102 citation statements)

References 36 publications

(44 reference statements)

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“…The expression levels measured by the cytochrome a/b ratio as shown in Zhen et al (22) ranged from 0.5 to 1.0. These enzymes were purified using nickel-nitrilotriacetic acid affinity column (see "Experimental Procedures").…”

Section: Design and Purification Of Mutantsmentioning

confidence: 94%

“…All of the mutated oxidases were constructed using the mutagenesis systems described (21). The mutated oxidases were overexpressed and purified using nickel-nitrilotriacetic acid affinity column chromatography as described by Zhen et al (22).…”

Section: Methodsmentioning

confidence: 99%

“…All of the mutants were successfully overexpressed using the system previously described (22). The expression levels measured by the cytochrome a/b ratio as shown in Zhen et al (22) ranged from 0.5 to 1.0.…”

Section: Design and Purification Of Mutantsmentioning

confidence: 99%

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Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

Zhen¹,

Hoganson²,

Babcock³

et al. 1999

Journal of Biological Chemistry

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Section: Design and Purification Of Mutantsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

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Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

Zhen¹,

Hoganson²,

Babcock³

et al. 1999

Journal of Biological Chemistry

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“…The CytcO was purified from the cell membranes using a Ni 2ϩ -NTA affinity column essentially as described in ref. 52.…”

Section: Methodsmentioning

confidence: 99%

A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase

Namslauer

Brzezinski

2009

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

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An increasing number of cancer types have been found to be linked to specific mutations in the mitochondrial DNA, which result in specific structural changes of the respiratory enzyme complexes. In this study, we have investigated the effect of 2 such mutations identified in colon cancer patients, leading to the amino acid substitutions Ser458Pro and Gly125Asp in subunit I of cytochrome c oxidase (complex IV) [Greaves et al. (2006) Proc Natl Acad Sci USA 103:714 -719]. We introduced these mutations in Rhodobacter sphaeroides, which carries an oxidase that serves as a model of the mitochondrial counterpart. The lack of expression of the former variant indicates that the amino acid substitution results in severely altered overall structure of the enzyme. The latter mutation (Gly171Asp in the bacterial oxidase) resulted in a structurally intact enzyme, but with reduced activity (approximately 30%), mainly due to slowed reduction of the redox site heme a. Furthermore, even though the Gly171Asp CytcO pumps protons, an intrinsic proton leak was identified, which would lead to a decreased overall energy-conversion efficiency of the respiratory chain, and would also perturb transport processes such as protein, ion, and metabolite trafficking. Furthermore, the specific leak may act to alter the balance between the electrical and chemical components of the proton electrochemical gradient. cytochrome aa3 ͉ electrochemical ͉ electron transfer ͉ pump ͉ respiratory oxidase

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“…27, and the enzyme was overexpressed and purified as described in Zhen et al (28). The position of M(II-263) is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Proton uptake controls electron transfer in cytochrome c oxidase

Karpefors

Ädelroth

Zhen

et al. 1998

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

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In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the ''peroxy'' to ''oxoferryl'' transition, internal transfer of the fourth electron from Cu A to heme a has the same rate as proton uptake from the bulk solution (8,000 s ؊1 ). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a Cu A ligand, was replaced by leucine, which resulted in an increased redox potential of Cu A . During reaction of the reduced mutant enzyme with O 2 , a proton was taken up at the same rate as in the wild-type enzyme (8,000 s ؊1 ), whereas electron transfer from Cu A to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from Cu A to heme a were blocked, the results from this study show that the Cu A 3 heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a 3 -Cu B before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.Cytochrome c oxidase is an integral membrane protein complex that catalyzes the sequential one-electron oxidations of cytochrome c coupled to the four-electron reduction of oxygen to water. Part of the free energy released in this process is conserved by translocation of protons across the membrane (for a recent review, see ref. 1). The coupling of electron transfer to proton transfer requires control of the rates of intramolecular electron-and proton-transfer reactions. To understand these physical processes on a molecular level, it is necessary to investigate in detail the individual electron-and proton-transfer steps during catalysis.The crystal structures of cytochrome c oxidase from bovine heart (2-4) and Paracoccus denitrificans (5, 6) have been determined to atomic resolution. The three-dimensional structure of the highly homologous Rhodobacter sphaeroides enzyme is generally assumed to be very similar to the structures of the P. denitrificans and bovine enzymes (7-9), and these structures are used as models of the R. sphaeroides enzyme. The R. sphaeroides cytochrome c oxidase consists of three protein subunits in which four redox-active metal sites are embedded. During enzyme turnover, electrons from watersoluble cytochrome c are transferred consecutively to Cu A

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Overexpression and Purification of CytochromecOxidase fromRhodobacter sphaeroides

Cited by 71 publications

References 36 publications

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase

Proton uptake controls electron transfer in cytochrome c oxidase

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