Electron transfer from cytochrome c(2) to the reaction center: a transition state model for ionic strength effects due to neutral mutations

Abstract

Interprotein electron transfer plays an important role in biological energy conversion. In this work, the electron transfer reaction between cytochrome c(2) (cyt) and the reaction center (RC) was studied to determine the mechanisms coupling association and electron transfer. Previous studies have shown that mutation of hydrophobic residues in the reaction interface, particularly Tyr L162, changes the binding affinity and rates of electron transfer at low ionic strengths. In this study, the effect of ionic strength on the second-order electron transfer rate constant, k(2), between cyt c(2) and native or mutant RCs was examined. Mutations of hydrophobic and hydrogen bonding residues caused k(2) to decrease more rapidly with an increase in ionic strength. This change is explained with a transition state model by a switch from a diffusion-limited reaction in native RCs, where electron transfer occurs upon each binding event, to a fast exchange reaction in the Tyr L162 mutant, where dissociation occurs before electron transfer and k(2) depends upon the equilibrium between bound and free protein complexes. The difference in ionic strength dependence is attributed to a smaller effect of ionic strength on the energy of the transition state compared to the bound state due to larger distances between charged residues in the transition state. This model explains the faster dissociation rate at higher ionic strengths that may assist rapid turnover that is important for biological function. These results provide a quantitative model for coupling protein association with electron transfer and elucidate the role of short-range interactions in determining the rate of electron transfer.

Figure 1

The structure of the cytochrome c₂:RC complex. The cofactors heme (Turquoise) on the cyt and bacteriochlorophyll dimer (Purple) on the RC are connected through a tightly packed interface region containing hydrophobic residues Tyr L162 and Leu M191 (Green) and hydrogen bonding residues Asn M187, Asn M188, and Gln L258(Yellow). Outside this tightly packed central region is a region of solvent separated complementary charged residues; negatively charged (Red) residues on the RC and positively charged (Blue) residues on the cyt (Axelrod et. al (9), PDB 1L9B).

Figure 2

Reaction coordinate diagram for association of the active cyt:RC complex for low ionic strength (solid line) and high ionic strength (dashed line). The energies of the separated proteins are set equal at the left. The change due to ionic strength of the rate of association k_on is dependent on the change in energy of the transition state ΔΔG^‡ while the change in the binding equilibrium constant K_A is dependent on the change in energy of the bound state ΔΔG. The parameter α is the ratio of the change in energy of the transition state ΔΔG^‡ and bound state ΔΔG due to ionic strength. (Modified from (12))

Figure 3

Transient absorbance changes due to electron transfer from cyt c₂ to RC following a laser flash. A) Native RCs showing biphasic kinetics characteristic of a diffusion limited reaction. The fast absorption change (τ =10⁻⁶ s) immediately following the laser flash due to reaction of bound cyt c₂ is not resolved in these traces.B) YA L162 RCs showing monophasic kinetics characteristic of a fast exchange reaction. The concentrations of the free cytochrome are as indicated. The concentration of RCs are 0.18 μM (native) and 0.46 μM (YA L162) (10 mM Hepes pH 7.5, 0.04% dodecyl-β-D-maltoside).

Figure 4

Plot of k₂ vs I^1/2 for native and mutant RCs. Native (■), LA M191 (◇), 3xHbond (▼), YA L162 (▲). The solid lines are fits to Eq. 9.

Figure 5

Ionic strength dependence of the binding constant K_A (■) and the second order rate constant k₂ (▲) for native RCs. The value of k_on is equal to that of k₂ assuming a diffusion limited reaction. The ratio of the slopes of log k_on and log K_A vs I^1/2 gives a value of α=0.60 ± 0.05 in good agreement with the value of α=0.59 ± 0.05 obtained from the fitting of the ionic strength dependence of k₂ in native and YA L162 RCs.

Figure 6

Ionic strength dependence of k_off and k_e for native RCs. The value of k_off was calculated from Eq.8. The value of k_off was always less than the value of k_e=10⁶ s⁻¹ justifying the assumption of a diffusion limited rate even at higher ionic strength.