Design of a Ruthenium-Cytochrome c Derivative To Measure Electron Transfer to the Radical Cation and Oxyferryl Heme in Cytochrome c Peroxidase
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- 作者：Kefei Wang ; Hongkang Mei ; Lois Geren ; Mark A. Miller ; Aleister Saunders ; Xuming Wang ; Jennifer L. Waldner ; Gary J. Pielak ; Bill Durham ; and Francis Millett
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：November 26, 1996
- 年：1996
- 卷：35
- 期：47
- 页码：15107 - 15119
- 全文大小：627K
- 年卷期：v.35,no.47(November 26, 1996)
- ISSN：1520-4995
文摘
A new ruthenium-labeled cytochrome c derivative wasdesigned to measure the actual rate ofelectron transfer to the Trp-191 radical cation and the oxyferryl hemein cytochrome c peroxidase compoundI{CMPI(FeIV=O,R
+)}.The H39C,C102T variant of yeast iso-1-cytochrome cwas labeled at the singlecysteine residue with a tris(bipyridyl)ruthenium(II)reagent to form Ru-39-Cc. This derivative has thesame reactivity with CMPI as native yCc measured by stopped-flowspectroscopy, indicating that theruthenium group does not interfere with the interaction between the twoproteins. Laser excitation of the1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mMphosphate, pH 7) resulted in electrontransfer from RuII* to heme c FeIIIwith a rate constant of 5 × 105s-1, followed by electron transferfromheme c FeII to the Trp-191 indolyl radicalcation inCMPI(FeIV=O,R+) with arate constant of keta =2× 106 s-1. A subsequentlaser flash led to electron transfer from heme c to theoxyferryl heme in CMPII(FeIV=O,R) with a rate constant ofketb = 5000s-1. The location of the binding domainwas determinedusing a series of surface charge mutants of CcP. The mutationsD34N, E290N, and A193F each decreasedthe values of keta andketb by 2-4-fold, consistent withthe use of the binding domain identified in thecrystal structure of the yCc-CcP complex for reduction of both redoxcenters [Pelletier, H., & Kraut, J.(1992) Science 258, 1748-1755]. A mechanism isproposed for reduction of the oxyferryl heme in whichinternal electron transfer in CMPII(FeIV=O,R) leadsto the regeneration of the radical cation in CMPII(FeIII,R+), which is thenreduced by yCcII. Thus, both steps in the completereduction of CMPI involveelectron transfer from yCcII to the Trp-191 radical cationusing the same binding site and pathway.Comparison of the rate constantketa with theoretical predictionsindicate that the electron transfer pathwayidentified in the crystalline yCc-CcP complex is very efficient.Stopped-flow studies indicate that nativeyCcII initially reduces the Trp-191 radical cation in CMPIwith a second-order rate constant ka,whichincreases from 1.8 × 108 M-1s-1 at 310 mM ionic strength to >3 ×109 M-1s-1 at ionic strengths below100 mM. A second molecule of yCcII then reduces theoxyferryl heme in CMPII with a second-orderrate constant kb which increases from 2.7 ×107 M-1s-1 at 310 mM ionic strength to 2.5 ×108 M-1s-1at 160 mM ionic strength. As the ionic strength is decreased below100 mM the rate constant for reductionof the oxyferryl heme becomes progressively slower as the reaction islimited by release of the productyCcIII from the yCcIII-CMPII complex.Both ruthenium photoreduction studies and stopped-flowstudiesdemonstrate that the Trp-191 radical cation is the initial site ofreduction in CMPI under all conditions ofionic strength.
+)}.The H39C,C102T variant of yeast iso-1-cytochrome cwas labeled at the singlecysteine residue with a tris(bipyridyl)ruthenium(II)reagent to form Ru-39-Cc. This derivative has thesame reactivity with CMPI as native yCc measured by stopped-flowspectroscopy, indicating that theruthenium group does not interfere with the interaction between the twoproteins. Laser excitation of the1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mMphosphate, pH 7) resulted in electrontransfer from RuII* to heme c FeIIIwith a rate constant of 5 × 105s-1, followed by electron transferfromheme c FeII to the Trp-191 indolyl radicalcation inCMPI(FeIV=O,R+) with arate constant of keta =2× 106 s-1. A subsequentlaser flash led to electron transfer from heme c to theoxyferryl heme in CMPII(FeIV=O,R) with a rate constant ofketb = 5000s-1. The location of the binding domainwas determinedusing a series of surface charge mutants of CcP. The mutationsD34N, E290N, and A193F each decreasedthe values of keta andketb by 2-4-fold, consistent withthe use of the binding domain identified in thecrystal structure of the yCc-CcP complex for reduction of both redoxcenters [Pelletier, H., & Kraut, J.(1992) Science 258, 1748-1755]. A mechanism isproposed for reduction of the oxyferryl heme in whichinternal electron transfer in CMPII(FeIV=O,R) leadsto the regeneration of the radical cation in CMPII(FeIII,R+), which is thenreduced by yCcII. Thus, both steps in the completereduction of CMPI involveelectron transfer from yCcII to the Trp-191 radical cationusing the same binding site and pathway.Comparison of the rate constantketa with theoretical predictionsindicate that the electron transfer pathwayidentified in the crystalline yCc-CcP complex is very efficient.Stopped-flow studies indicate that nativeyCcII initially reduces the Trp-191 radical cation in CMPIwith a second-order rate constant ka,whichincreases from 1.8 × 108 M-1s-1 at 310 mM ionic strength to >3 ×109 M-1s-1 at ionic strengths below100 mM. A second molecule of yCcII then reduces theoxyferryl heme in CMPII with a second-orderrate constant kb which increases from 2.7 ×107 M-1s-1 at 310 mM ionic strength to 2.5 ×108 M-1s-1at 160 mM ionic strength. As the ionic strength is decreased below100 mM the rate constant for reductionof the oxyferryl heme becomes progressively slower as the reaction islimited by release of the productyCcIII from the yCcIII-CMPII complex.Both ruthenium photoreduction studies and stopped-flowstudiesdemonstrate that the Trp-191 radical cation is the initial site ofreduction in CMPI under all conditions ofionic strength.