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Visualizing changes in electron distribution in coupled chains of cytochrome bc(1) by modifying barrier for electron transfer between the FeS cluster and heme c(1).

Cieluch E, Pietryga K, Sarewicz M, Osyczka A - Biochim. Biophys. Acta (2009)

Bottom Line: This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide.In the non-inhibited system no such differences were observed.We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-307 Kraków, Poland.

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Analysis of post-flash electron distribution in wild type and A181T cytochrome bc1. Schemes in A, B compare wild type (WT) with A181T for a given set of conditions. Black and white squares represent reduced and oxidized cofactors, respectively. Incomplete reduction is represented in a grey scale. Dotted squares mark the cofactors of c-chain that are observable in the flash experiments. (A) Single flash generates two oxidizing equivalents per cytochrome bc1 (white squares c) [38] at the time where hemes b are oxidized (white squares bL and bH) and the FeS cluster and heme c1 are reduced (black squares FeS and c1, respectively). (B) Within tens of milliseconds electrons redistribute to reach equilibrium in which the reduction levels of cofactors vary depending on experimental conditions. In the absence of inhibitors, unperturbed electron flow out of the b-chain upon oxidation of two QH2 secures complete re-reduction of all cofactors in the c-chain in both WT and A181T (black squares in no inhibitor panel). In the presence of antimycin, the level of reduced hemes b available for reverse reaction is higher and the equilibrium is reached before the c-chain is fully reduced. In WT, incomplete oxidation of second QH2 (grey squares in antimycin panel) leaves FeS partially oxidized, which leads to redistribution of electrons in the entire c-chain, as observed by flash at the level of cytochromes c (note that intensity of squares in c-chain should be reduced, which for simplicity is not shown). In A181T, incomplete oxidation of second QH2 faces additional barrier of potential difference between FeS and heme c1 which shifts equilibrium toward reduced FeS at the expense of reduced heme c1 (represented as black square FeS and light grey square c1). This increases probability of reverse reaction and decreases probability of forward reaction at the Qo site, and the level of reduced heme bL may decrease (note that in this case the reduction level of heme c1 determines the reduction level of heme bL, as represented by light grey squares c1 and bL). In the presence of myxothiazol (myxothiazol panel), electron from pre-reduced FeS cluster redistributes among the cofactors of the c-chain, but in WT the oxidation of FeS cluster by cytochromes c is more prominent than in A181T (note that for simplicity, for WT a complete electron transfer from FeS cluster to heme c is shown, white square FeS and black squares c). In the presence of stigmatellin (stigmatellin panel) only cytochromes c are in equilibrium thus full extent of flash-oxidized cytochromes c is preserved. Panel (C) shows simulations of the traces for the reduction of the observable experimentally cytochromes c in WT (dashed line) and A181T (solid line) obtained from the model schematically presented in panel (D). The time constants for partial reactions denote k0f/k0b — association/dissociation rate of Q to/from the Qo site, k1f/k1b — same as k0f/k0b but for QH2, k2f/k2b — two-electron oxidation/reduction of QH2/Q in the Qo site, k3f/k3b — rate constants for movement of the FeS head domain to/from cytochrome c1 position, k4f/k4b — rate constants for electron transfer from FeS to heme c1 or reverse reaction, k5f/k5b — rate constant for electron transfer from heme c1 to heme c or reverse reaction. Dotted line in the antimycin panel in C shows the trace simulated for A181T assuming that QH2 oxidation at the Qo site was concerted but irreversible (i.e., k2b = 0 M-1s-1). This illustrates the prediction of how the system would respond if at least one reaction was irreversible (a case not supported experimentally).
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fig6: Analysis of post-flash electron distribution in wild type and A181T cytochrome bc1. Schemes in A, B compare wild type (WT) with A181T for a given set of conditions. Black and white squares represent reduced and oxidized cofactors, respectively. Incomplete reduction is represented in a grey scale. Dotted squares mark the cofactors of c-chain that are observable in the flash experiments. (A) Single flash generates two oxidizing equivalents per cytochrome bc1 (white squares c) [38] at the time where hemes b are oxidized (white squares bL and bH) and the FeS cluster and heme c1 are reduced (black squares FeS and c1, respectively). (B) Within tens of milliseconds electrons redistribute to reach equilibrium in which the reduction levels of cofactors vary depending on experimental conditions. In the absence of inhibitors, unperturbed electron flow out of the b-chain upon oxidation of two QH2 secures complete re-reduction of all cofactors in the c-chain in both WT and A181T (black squares in no inhibitor panel). In the presence of antimycin, the level of reduced hemes b available for reverse reaction is higher and the equilibrium is reached before the c-chain is fully reduced. In WT, incomplete oxidation of second QH2 (grey squares in antimycin panel) leaves FeS partially oxidized, which leads to redistribution of electrons in the entire c-chain, as observed by flash at the level of cytochromes c (note that intensity of squares in c-chain should be reduced, which for simplicity is not shown). In A181T, incomplete oxidation of second QH2 faces additional barrier of potential difference between FeS and heme c1 which shifts equilibrium toward reduced FeS at the expense of reduced heme c1 (represented as black square FeS and light grey square c1). This increases probability of reverse reaction and decreases probability of forward reaction at the Qo site, and the level of reduced heme bL may decrease (note that in this case the reduction level of heme c1 determines the reduction level of heme bL, as represented by light grey squares c1 and bL). In the presence of myxothiazol (myxothiazol panel), electron from pre-reduced FeS cluster redistributes among the cofactors of the c-chain, but in WT the oxidation of FeS cluster by cytochromes c is more prominent than in A181T (note that for simplicity, for WT a complete electron transfer from FeS cluster to heme c is shown, white square FeS and black squares c). In the presence of stigmatellin (stigmatellin panel) only cytochromes c are in equilibrium thus full extent of flash-oxidized cytochromes c is preserved. Panel (C) shows simulations of the traces for the reduction of the observable experimentally cytochromes c in WT (dashed line) and A181T (solid line) obtained from the model schematically presented in panel (D). The time constants for partial reactions denote k0f/k0b — association/dissociation rate of Q to/from the Qo site, k1f/k1b — same as k0f/k0b but for QH2, k2f/k2b — two-electron oxidation/reduction of QH2/Q in the Qo site, k3f/k3b — rate constants for movement of the FeS head domain to/from cytochrome c1 position, k4f/k4b — rate constants for electron transfer from FeS to heme c1 or reverse reaction, k5f/k5b — rate constant for electron transfer from heme c1 to heme c or reverse reaction. Dotted line in the antimycin panel in C shows the trace simulated for A181T assuming that QH2 oxidation at the Qo site was concerted but irreversible (i.e., k2b = 0 M-1s-1). This illustrates the prediction of how the system would respond if at least one reaction was irreversible (a case not supported experimentally).

Mentions: The full cytochrome c re-reduction in the absence of any inhibitor consumes electrons from the pre-reduced FeS cluster and then from quinols that are oxidized at the Qo site in a coupled reaction that delivers the second quinol-born electron to the b-chain. A completeness of this reaction is a consequence of the unperturbed outflow of electrons from hemes b to the Qi site and further down to the Q pool. In another words, a completion of two reactions of QH2 oxidation at the Qo site secured by an immediate removal of electrons from hemes b leaves all cofactors in the c-chain (including the FeS cluster) fully saturated with electrons. The reaction is driven to completion even when heme c1 has potential 100 mV lower than Em of FeS in the mutant (Fig. 6, no inhibitor panel). This is consistent with the observations that the Em of heme c1 can be lowered within this range without affecting the overall electron flow through the c-chain [27,44]. In fact, the difference in Ems between heme c1 and the FeS cluster can be as much as 180 mV and the enzyme still muster enough electron transfer through cytochrome bc1 to support its functionality [27].


Visualizing changes in electron distribution in coupled chains of cytochrome bc(1) by modifying barrier for electron transfer between the FeS cluster and heme c(1).

Cieluch E, Pietryga K, Sarewicz M, Osyczka A - Biochim. Biophys. Acta (2009)

Analysis of post-flash electron distribution in wild type and A181T cytochrome bc1. Schemes in A, B compare wild type (WT) with A181T for a given set of conditions. Black and white squares represent reduced and oxidized cofactors, respectively. Incomplete reduction is represented in a grey scale. Dotted squares mark the cofactors of c-chain that are observable in the flash experiments. (A) Single flash generates two oxidizing equivalents per cytochrome bc1 (white squares c) [38] at the time where hemes b are oxidized (white squares bL and bH) and the FeS cluster and heme c1 are reduced (black squares FeS and c1, respectively). (B) Within tens of milliseconds electrons redistribute to reach equilibrium in which the reduction levels of cofactors vary depending on experimental conditions. In the absence of inhibitors, unperturbed electron flow out of the b-chain upon oxidation of two QH2 secures complete re-reduction of all cofactors in the c-chain in both WT and A181T (black squares in no inhibitor panel). In the presence of antimycin, the level of reduced hemes b available for reverse reaction is higher and the equilibrium is reached before the c-chain is fully reduced. In WT, incomplete oxidation of second QH2 (grey squares in antimycin panel) leaves FeS partially oxidized, which leads to redistribution of electrons in the entire c-chain, as observed by flash at the level of cytochromes c (note that intensity of squares in c-chain should be reduced, which for simplicity is not shown). In A181T, incomplete oxidation of second QH2 faces additional barrier of potential difference between FeS and heme c1 which shifts equilibrium toward reduced FeS at the expense of reduced heme c1 (represented as black square FeS and light grey square c1). This increases probability of reverse reaction and decreases probability of forward reaction at the Qo site, and the level of reduced heme bL may decrease (note that in this case the reduction level of heme c1 determines the reduction level of heme bL, as represented by light grey squares c1 and bL). In the presence of myxothiazol (myxothiazol panel), electron from pre-reduced FeS cluster redistributes among the cofactors of the c-chain, but in WT the oxidation of FeS cluster by cytochromes c is more prominent than in A181T (note that for simplicity, for WT a complete electron transfer from FeS cluster to heme c is shown, white square FeS and black squares c). In the presence of stigmatellin (stigmatellin panel) only cytochromes c are in equilibrium thus full extent of flash-oxidized cytochromes c is preserved. Panel (C) shows simulations of the traces for the reduction of the observable experimentally cytochromes c in WT (dashed line) and A181T (solid line) obtained from the model schematically presented in panel (D). The time constants for partial reactions denote k0f/k0b — association/dissociation rate of Q to/from the Qo site, k1f/k1b — same as k0f/k0b but for QH2, k2f/k2b — two-electron oxidation/reduction of QH2/Q in the Qo site, k3f/k3b — rate constants for movement of the FeS head domain to/from cytochrome c1 position, k4f/k4b — rate constants for electron transfer from FeS to heme c1 or reverse reaction, k5f/k5b — rate constant for electron transfer from heme c1 to heme c or reverse reaction. Dotted line in the antimycin panel in C shows the trace simulated for A181T assuming that QH2 oxidation at the Qo site was concerted but irreversible (i.e., k2b = 0 M-1s-1). This illustrates the prediction of how the system would respond if at least one reaction was irreversible (a case not supported experimentally).
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fig6: Analysis of post-flash electron distribution in wild type and A181T cytochrome bc1. Schemes in A, B compare wild type (WT) with A181T for a given set of conditions. Black and white squares represent reduced and oxidized cofactors, respectively. Incomplete reduction is represented in a grey scale. Dotted squares mark the cofactors of c-chain that are observable in the flash experiments. (A) Single flash generates two oxidizing equivalents per cytochrome bc1 (white squares c) [38] at the time where hemes b are oxidized (white squares bL and bH) and the FeS cluster and heme c1 are reduced (black squares FeS and c1, respectively). (B) Within tens of milliseconds electrons redistribute to reach equilibrium in which the reduction levels of cofactors vary depending on experimental conditions. In the absence of inhibitors, unperturbed electron flow out of the b-chain upon oxidation of two QH2 secures complete re-reduction of all cofactors in the c-chain in both WT and A181T (black squares in no inhibitor panel). In the presence of antimycin, the level of reduced hemes b available for reverse reaction is higher and the equilibrium is reached before the c-chain is fully reduced. In WT, incomplete oxidation of second QH2 (grey squares in antimycin panel) leaves FeS partially oxidized, which leads to redistribution of electrons in the entire c-chain, as observed by flash at the level of cytochromes c (note that intensity of squares in c-chain should be reduced, which for simplicity is not shown). In A181T, incomplete oxidation of second QH2 faces additional barrier of potential difference between FeS and heme c1 which shifts equilibrium toward reduced FeS at the expense of reduced heme c1 (represented as black square FeS and light grey square c1). This increases probability of reverse reaction and decreases probability of forward reaction at the Qo site, and the level of reduced heme bL may decrease (note that in this case the reduction level of heme c1 determines the reduction level of heme bL, as represented by light grey squares c1 and bL). In the presence of myxothiazol (myxothiazol panel), electron from pre-reduced FeS cluster redistributes among the cofactors of the c-chain, but in WT the oxidation of FeS cluster by cytochromes c is more prominent than in A181T (note that for simplicity, for WT a complete electron transfer from FeS cluster to heme c is shown, white square FeS and black squares c). In the presence of stigmatellin (stigmatellin panel) only cytochromes c are in equilibrium thus full extent of flash-oxidized cytochromes c is preserved. Panel (C) shows simulations of the traces for the reduction of the observable experimentally cytochromes c in WT (dashed line) and A181T (solid line) obtained from the model schematically presented in panel (D). The time constants for partial reactions denote k0f/k0b — association/dissociation rate of Q to/from the Qo site, k1f/k1b — same as k0f/k0b but for QH2, k2f/k2b — two-electron oxidation/reduction of QH2/Q in the Qo site, k3f/k3b — rate constants for movement of the FeS head domain to/from cytochrome c1 position, k4f/k4b — rate constants for electron transfer from FeS to heme c1 or reverse reaction, k5f/k5b — rate constant for electron transfer from heme c1 to heme c or reverse reaction. Dotted line in the antimycin panel in C shows the trace simulated for A181T assuming that QH2 oxidation at the Qo site was concerted but irreversible (i.e., k2b = 0 M-1s-1). This illustrates the prediction of how the system would respond if at least one reaction was irreversible (a case not supported experimentally).
Mentions: The full cytochrome c re-reduction in the absence of any inhibitor consumes electrons from the pre-reduced FeS cluster and then from quinols that are oxidized at the Qo site in a coupled reaction that delivers the second quinol-born electron to the b-chain. A completeness of this reaction is a consequence of the unperturbed outflow of electrons from hemes b to the Qi site and further down to the Q pool. In another words, a completion of two reactions of QH2 oxidation at the Qo site secured by an immediate removal of electrons from hemes b leaves all cofactors in the c-chain (including the FeS cluster) fully saturated with electrons. The reaction is driven to completion even when heme c1 has potential 100 mV lower than Em of FeS in the mutant (Fig. 6, no inhibitor panel). This is consistent with the observations that the Em of heme c1 can be lowered within this range without affecting the overall electron flow through the c-chain [27,44]. In fact, the difference in Ems between heme c1 and the FeS cluster can be as much as 180 mV and the enzyme still muster enough electron transfer through cytochrome bc1 to support its functionality [27].

Bottom Line: This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide.In the non-inhibited system no such differences were observed.We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-307 Kraków, Poland.

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Related in: MedlinePlus