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Kinetic studies of the reaction of c...

University of Arkansas.

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Kinetic studies of the reaction of cytochrome C with cytochrome C oxidase.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Kinetic studies of the reaction of cytochrome C with cytochrome C oxidase./
作者:	Brand, Sue Ellen.
面頁冊數:	189 p.
附註:	Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2971.
Contained By:	Dissertation Abstracts International69-05B.
標題:	Biophysics, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3317798
ISBN:	9780549661108

Kinetic studies of the reaction of cytochrome C with cytochrome C oxidase.
Brand, Sue Ellen.

Kinetic studies of the reaction of cytochrome C with cytochrome C oxidase. - 189 p.

Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2971.

Thesis (Ph.D.)--University of Arkansas, 2008.

The first two steps (OH → EH and EH → PM transitions) in the reductive portion of the catalytic cycle of cytochrome c oxidase (CcO) were studied using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine) 2]2(quaterpyridine), (Ru2Z). The ultimate goal of this study was to determine whether the redox kinetics were affected by pulsing (i.e., fully reducing the enzyme prior to reoxidation) CcO before photoreduction. Previous studies reported changes in the redox kinetics with the pulsing procedure. The new photoreductant has a large quantium yield (60% reduction for CuA), allowing for the kinetics to be monitored with a single flash. The +4 charge on Ru2Z allows electrostatic binding to occur on subunit II near the CuA site of CcO. A flow flash technique (stopped-flow coupled to flash photolysis) was used to generate the pulsed form of CcO (the "OH" state) from several species including Bos taurus (bovine), Rhodobacter sphaeroides (R.s) and Paracoccus denitrificans (P.d). Electron transfer was monitored spectroscopically from CuA→heme a→heme a3/CuB in CcO and results were compared to the non-pulsed enzyme. Pulsing had a significant effect on the kinetics for the bovine oxidase but had no effect for the bacterial enzymes. The electron transfer rate constant for the reduction of heme a was 20,000 s-1, and was independent of sample preparation. However, electron transfer from heme a to the binuclear center (heme a3 and CuB) varied due to sample preparation. Oxidation of heme a occurred with 63% completeness and biphasic kinetics with rate constants of 750 s-1 and 110 s-l and amplitudes of 25% and 75% were observed. The non-pulsed bovine enzyme demonstrated monophasic kinetics with a rate constant of 90s-1 and an amplitude of 30%. This is the first evidence that the pulsing procedure can produce different results in the fast form of bovine CcO. Reduction of heme a3 was not observed indicating that CuB may be the initial electron acceptor in the binuclear site. The second electron into CcO was measured by first pulsing CcO to generate the OH state then one-electron reduction of CcO was accomplished using reduced K13E cytochrome c (Cc). The K13E Cc mutant was specifically designed to rapidly reduce CcO generating the EH state then photoreduction of the Ru2Z complex provided the second electron to CcO. Biphasic kinetics were observed for the oxidation of heme a with rate constants of 1100 s-1 and 90 s-1 with amplitudes of 11% and 89%. The formation of state P was indicated by the previously reported rate constant of 90s-1.

ISBN: 9780549661108Subjects--Topical Terms:

1019105
Biophysics, General.

Kinetic studies of the reaction of cytochrome C with cytochrome C oxidase.
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100 1 $a Brand, Sue Ellen. $3 1030678
245 1 0 $a Kinetic studies of the reaction of cytochrome C with cytochrome C oxidase.
300 $a 189 p.
500 $a Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2971.
502 $a Thesis (Ph.D.)--University of Arkansas, 2008.
520 $a The first two steps (OH → EH and EH → PM transitions) in the reductive portion of the catalytic cycle of cytochrome c oxidase (CcO) were studied using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine) 2]2(quaterpyridine), (Ru2Z). The ultimate goal of this study was to determine whether the redox kinetics were affected by pulsing (i.e., fully reducing the enzyme prior to reoxidation) CcO before photoreduction. Previous studies reported changes in the redox kinetics with the pulsing procedure. The new photoreductant has a large quantium yield (60% reduction for CuA), allowing for the kinetics to be monitored with a single flash. The +4 charge on Ru2Z allows electrostatic binding to occur on subunit II near the CuA site of CcO. A flow flash technique (stopped-flow coupled to flash photolysis) was used to generate the pulsed form of CcO (the "OH" state) from several species including Bos taurus (bovine), Rhodobacter sphaeroides (R.s) and Paracoccus denitrificans (P.d). Electron transfer was monitored spectroscopically from CuA→heme a→heme a3/CuB in CcO and results were compared to the non-pulsed enzyme. Pulsing had a significant effect on the kinetics for the bovine oxidase but had no effect for the bacterial enzymes. The electron transfer rate constant for the reduction of heme a was 20,000 s-1, and was independent of sample preparation. However, electron transfer from heme a to the binuclear center (heme a3 and CuB) varied due to sample preparation. Oxidation of heme a occurred with 63% completeness and biphasic kinetics with rate constants of 750 s-1 and 110 s-l and amplitudes of 25% and 75% were observed. The non-pulsed bovine enzyme demonstrated monophasic kinetics with a rate constant of 90s-1 and an amplitude of 30%. This is the first evidence that the pulsing procedure can produce different results in the fast form of bovine CcO. Reduction of heme a3 was not observed indicating that CuB may be the initial electron acceptor in the binuclear site. The second electron into CcO was measured by first pulsing CcO to generate the OH state then one-electron reduction of CcO was accomplished using reduced K13E cytochrome c (Cc). The K13E Cc mutant was specifically designed to rapidly reduce CcO generating the EH state then photoreduction of the Ru2Z complex provided the second electron to CcO. Biphasic kinetics were observed for the oxidation of heme a with rate constants of 1100 s-1 and 90 s-1 with amplitudes of 11% and 89%. The formation of state P was indicated by the previously reported rate constant of 90s-1.
520 $a The role of a second Cc binding site (low affinity site) was studied. A ruthenium complex (Ru(bpy)2(mobpy)) covalently bound to horse Cc at Cys-39 (Ru-39-Cc) was used to initiate electron transfer to native bovine cytochrome oxidase (CcO). A slow phase rate constant of 200 s-1 was observed for 2:1 ratios of Ru-39-Cc to bovine CcO. Increasing oxidized native horse Cc, which acts as a competitive inhibitor, had no effect on the slow rate of bovine CcO at 0 mM NaCl. A three-binding site model was made to explain the unusual kinetics. Increasing the ionic strength proved to decrease the binding affinity so that only two Cc binding site were measured at 40 mM NaCl and only one binding site was measured at 70 mM NaCl. Analytical ultracentrifugation confirmed the binding behavior for bovine CcO. Experiments were repeated for R.s CcO and three binding sites were also measured. The role of the low affinity site appears to be to increase product dissociation at the high affinity site for R.s CcO. The role of the low affinity site for bovine CcO is unknown. (Abstract shortened by UMI.)
590 $a School code: 0011.
650 4 $a Biophysics, General. $3 1019105
650 4 $a Chemistry, Biochemistry. $3 1017722
650 4 $a Chemistry, Physical. $3 560527
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690 $a 0494
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710 2 $a University of Arkansas. $3 1017562
773 0 $t Dissertation Abstracts International $g 69-05B.
790 $a 0011
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792 $a 2008
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