An Arginine to Lysine Mutation in the Vicinity of the Heme Propionates Affects the Redox Potentials of the Hemes and Associated Electron and Proton Transfer in Cytochrome c Oxidase
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- 作者：Denise A. Mills ; Lois Geren ; Carrie Hiser ; Bryan Schmidt ; Bill Durham ; Francis Millett ; and Shelagh Ferguson-Miller
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 9, 2005
- 年：2005
- 卷：44
- 期：31
- 页码：10457 - 10465
- 全文大小：252K
- 年卷期：v.44,no.31(August 9, 2005)
- ISSN：1520-4995
文摘
Cytochrome c oxidase pumps protons across a membrane using energy from electron transferand reduction of oxygen to water. It is postulated that an element of the energy transduction mechanismis the movement of protons to the vicinity of the hemes upon reduction, to favor charge neutrality. Possiblesites on which protons could reside, in addition to the conserved carboxylate E286, are the propionategroups of heme a and/or heme a3. A highly conserved pair of arginines (R481 and R482) interact withthese propionates through ionic and hydrogen bonds. This study shows that the conservative mutant,R481K, although as fully active as the wild type under many conditions, exhibits a significant decreasein the midpoint redox potential of heme a relative to CuA (
Em) of 
40 mV, has lowered activity underconditions of high pH or in the presence of a membrane potential, and has a slowed heme a3 reductionwith dithionite. Another mutant, D132A, which strongly inhibits proton uptake from the internal side ofthe membrane, has <4% of the activity of the wild type and appears to be dependent on proton uptakefrom the outside. A double mutation, D132A/R481K, is even more strongly inhibited (~1% of that of thewild type). The more-than-additive effect supports the concept that R481K not only lowers the midpointpotential of heme a but also limits a supply route for protons from the outside of the membrane used bythe D132 mutant. The results are consistent with an important role of R481 and heme a/a3 propionates inproton movement in a reversible exit path.
Em) of 40 mV, has lowered activity underconditions of high pH or in the presence of a membrane potential, and has a slowed heme a3 reductionwith dithionite. Another mutant, D132A, which strongly inhibits proton uptake from the internal side ofthe membrane, has <4% of the activity of the wild type and appears to be dependent on proton uptakefrom the outside. A double mutation, D132A/R481K, is even more strongly inhibited (~1% of that of thewild type). The more-than-additive effect supports the concept that R481K not only lowers the midpointpotential of heme a but also limits a supply route for protons from the outside of the membrane used bythe D132 mutant. The results are consistent with an important role of R481 and heme a/a3 propionates inproton movement in a reversible exit path.