Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site

Abstract

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redoxp roteins is often optimizedt os teer ar edox partner to the electron transfer active binding site.T ot est whether the optimizeddistribution is more important than the strength of the electrostatic interactions,anadditional negative patch was introduced on the surface of cytochrome cp eroxidase,a way from the stereospecific binding site,a nd its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner,c ytochrome c, interacts with the new patch.U nexpectedly,t he rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimizedc harge distribution.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed

“… 30 CcP_B additionally has the K21E, K29E, K90E, and K97E mutations. 27 CcP_C contains the same mutations as CcP_B and D35N, E36Q, E119Q, E202Q, E210Q, E215Q, D225N, and E291Q that neutralize the charges of the WT binding site. CcP_D has mutations K75E, K76Q, N79D, Q87E, K91Q, K98Q, and N142D in comparison to CcP_A.…”

“…The structures were prepared, and the Monte Carlo docking simulations were performed as described in ref ( 27 ). The structures of CcP and Cc from Protein Data Bank (PDB) entry 2PCC( 16 ) were used as a basis for the simulations.…”

“…The results of the kinetics measured for the Cc:CcP_A and Cc:CcP_B complexes at ionic strengths from 752 to 122 mM were presented in our previous work. 27 The same protocols were followed to measure the reactions between Cc and the other mutants of CcP. The kinetic measurements were performed at ionic strengths 752, 622, 502, 392, 292, 192, 122, and 44 mM on a SX20 stopped flow spectrometer (AppliedPhotophysics, Leatherhead, Surrey, U.K.) with a 1.2 ms dead time at room temperature following the absorbance at 416 nm, which indicates oxidation of Cc.…”

“…The stopped flow data were fitted to eq 1 as previously described: 27 where B 0 is the starting concentration of Cc(Fe 2+ ) and CpdI after mixing, Δε 416 is the difference in extinction coefficient at 416 nm for oxidized and reduced Cc, −40 mM –1 cm –1 , 40 − 42 k obs (inverse seconds) is the observed rate constant of reduction, t is the time (seconds), and C corrects for the baseline voltage of the spectrometer.…”

“…In a study from our groups, the charge distribution on the surface of CcP was changed strongly by addition of a negative patch on one side of CcP. 27 Monte Carlo simulations and paramagnetic relaxation enhancement (PRE) experiments showed that Cc interacts with the added patch, enlarging the surface visited in the encounter complex. The added charges also slightly enhance k a .…”