Mutants of the Cu_A Site in Cytochrome c Oxidase of Rhodobacter sphaeroides:  II. Rapid Kinetic Analysis of Electron Transfer

Abstract

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.

The electron transfer complex between nitrous oxide reductase and its electron donors

“…5b-pathway represented in blue). This electron transfer route is analogous to that proposed for the electron transfer from the CuA center to the heme a in cytochrome c oxidase [56].…”

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

“…The ionic strength of the aqueous medium was set to 75 mM KCl (pH 8.0) where the complex of horse heart cyt c and bacterial CcO exhibits the most efficient electron-transfer kinetics. 45 The cyclic voltammogram (Figure 9) demonstrates the reversible redox reaction between cyt c and the surface-tethered CcO. The midpoint potential is 244.1 mV vs NHE as determined by a faster scan rate of 50 mV/s (data not shown).…”

Biological inorganic chemistry at the beginning of the 21st century

“…uring June [16][17][18][19][20]1976, several hundred chemists and biologists assembled at the University of British Columbia (UBC) to listen to 13 lectures and discuss recent developments at the interface of inorganic chemistry and biology. To be sure, there had been many previous meetings at which this new science was featured, notably one in Blacksburg, Virginia (1), and several others on special topics that were held during the 1950s and 1960s.…”

“…1; refs. [8][9][10][11][12][13][14]; the coupling of cytochrome c to Cu A (15)(16)(17), as well as Cu A to cytochrome a (18,19); and the redox activity, if any, during turnover, of the covalently coupled tyrosine-histidine copper ligand in the binuclear heme a 3 -Cu B active site (20). In this issue of PNAS, both Ken Karlin and Yi Lu report experiments that shed light on intermediates that likely are involved in the course of dioxygen activation͞reduction by hemecopper pairs (21,22), and Bill Tolman contributes to the discussion of dioxygen activation by metals in his density functional theoretical (DFT) analysis of the extent of electron transfer in side-bonded complexes (23).…”