Comparison of Dicyclohexylcarbodiimide (DCCD)-Induced Effects on Structure and Activity in Cytochrome c Oxidase (COX) from Bovine Heart and Rhodobacter Sphaeroides

Abstract

Previous work has shown that DCCD inhibits bovine heart cytochrome c oxidase (COX) function by binding to Glu-90 in subunit III (SIII). The goal of this study was to compare the effects of DCCD modification on structure and electron transfer in COX from bovine heart and Rhodobacter sphaeroides. DCCD modification induced similar shifts in migration of SIII on SDS-PAGE in both enzymes, suggesting similar SIII modification. The concentration dependence for the inhibition of electron transfer by DCCD was also similar in both enzymes. The pH dependence of electron transfer activity in the DCCD-modified enzymes was acid shifted 0.7-0.9 pH unit in both enzymes. However, bovine heart COX treated with DCCD exhibited less inhibition of electron transfer activity at pH 9.5-10.0 than at lower pH, while DCCD treated R. sphaeroides COX had less inhibition at pH 6.5-7.0 than at higher pH. The heme circular dichroism spectrum showed that DCCD induced a red shift of 0.5-0.8 nm in both enzymes when monitored at both pH 7.0 and 10.0. Steady-state heme a reduction during electron transfer increased from 18±1% to 33±2% in DCCD-modified beef COX at pH 7.0, while in R. sphaeroides, it remained unchanged from 41±2% to 36±2%. In summary, our results indicate that DCCD, while binding at a similar site in SIII, leads to differential effects in the two enzymes. DCCD modification of SIII of bovine heart may cause blockage of the putative O2 transfer pathway, while in R. sphaeroides it may induce a slowed proton uptake. The different characteristics of inhibition may be due to variation in both the number of subunits and their structural homology between the two forms of COX forms.