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Design of a Ruthenium−Cytochrome c Derivative To Measure Electron Transfer to the Radical Cation and Oxyferryl Heme in Cytochrome c Peroxidase
Journal article   Peer reviewed

Design of a Ruthenium−Cytochrome c Derivative To Measure Electron Transfer to the Radical Cation and Oxyferryl Heme in Cytochrome c Peroxidase

Kefei Wang, Hongkang Mei, Lois Geren, Mark A. Miller, Aleister Saunders, Xuming Wang, Jennifer L. Waldner, Gary J. Pielak, Bill Durham and Francis Millett
Biochemistry (Easton), v 35(47), pp 15107-15119
26 Nov 1996
DOI: https://doi.org/10.1021/bi9611117
PMID: 8942678
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIVO,R•+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris(bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc−CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 × 105 s-1, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation in CMPI(FeIVO,R•+) with a rate constant of keta = 2 × 106 s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII(FeIVO,R) with a rate constant of ketb = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of keta and ketb by 2−4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc−CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748−1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIVO,R) leads to the regeneration of the radical cation in CMPII(FeIII,R•+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant keta with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc−CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 × 108 M-1 s-1 at 310 mM ionic strength to >3 × 109 M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 × 107 M-1 s-1 at 310 mM ionic strength to 2.5 × 108 M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII−CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.

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