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In the cell metabolic cycle, cytochrome c oxidase (COX) is the final electron acceptor of the respiratory chain which reduces molecular oxygen into water. It is bound in the inner mitochondrial membrane and on the plasma membrane of bacterial species. Energy produced through electron transfer is coupled to the pumping of protons against the electrochemical gradient in order to fuel the proton motive force for the synthesis of most of the ATP in the cell. The three mitochondrial encoded subunits of COX, I, II, and III, are conserved across species and the complete function of subunit III remains unknown. Dicyclohexylcarbodiimide (DCCD) is an inhibitor of function which binds specifically to the conserved Glu-90 residue of subunit III. DCCD modification of COX has been shown to induce a conformational change in subunit III, which inhibits the proton pumping and electron transfer mechanisms occurring in subunit I of the enzyme.This work analyzes the catalytic mechanism and environment of bovine heart and R. sphaeroides COX upon DCCD modification in order to gain insight into the significance of subunit III in the functioning of the enzyme as well as to compare the effects of the modification on catalytic activity. The effect of DCCD modification was also analyzed in both physiological and alkaline environments due to data which showed that bovine heart COX exhibits less inhibition of electron transfer activity at pH values 9.5 and 10.0, while R. sphaeroides COX shows less inhibition at pH values 6.5 and 7.0. Both COX enzymes exhibited a steady biphasic pH dependence for electron transfer activity, suggesting that there are two proton binding sites critical in electron transfer activity. Bovine heart COX displayed an alkaline shift from 8.8±0.2 to 9.3±0.1 at site 2, while R. sphareoides COX displayed an acidic shift from 7.8±0.4 to 7.3±0.4 at site 1. To examine the effects of DCCD modification on the environment of hemes a and a3, the Soret region of the CD spectrum was analyzed. DCCD induced a red shift from 427.7 ± 0.3 nm in control to 428.2 ± 0.1 nm at pH 7.0 in bovine heart COX and from 429.2 ± 0.2 nm to 429.6 ± 0.1 nm at pH 10. In R. sphaeroides, a red shift in the CD spectrum was observed from 429.4 ± 0.1 nm in WT to 430.2 ± 0.1 nm in the DCCD-modified enzyme at pH 7.0 and from 431.5 ± 0.5 nm in WT to 431.9 ± 0.4 nm in DCCD-modified enzyme at pH 10.0. The heme a and a3 environment was also monitored using heme a reduction during steady state electron transfer. Heme a was found to be 18±1% reduced in control bovine heart COX during electron transfer and 33±2% reduced in DCCD-modified COX at pH 7.0 At pH 10.0, control bovine heart COX exhibited a heme a reduction level of 67±7% and DCCD-modified enzyme yielded a reduction level of 93±6%. In0 R. sphaeroides, heme a was found to be 41 ± 2% reduced and DCCD-modified enzyme was 36 ± 2% reduced at pH 7.0. At pH 10.0, WT R. sphaeroides exhibited a heme a reduction level of 33 ± 4% and DCCD-modified enzyme yielded a reduction level of 47 ± 5%. In summary, our results indicate that the modification at Glu-90 in subunit III causes a perturbation to the catalytic cycle and its environment in subunit I. DCCD, while binding at a similar site in subunit III, leads to differential effects on the conformation and activity of the enzyme in bovine heart and R. sphaeroides COX. DCCD modification to subunit III of bovine heart COX may cause blockage of the putative O2 transfer pathway, while modification of subunit III in R. sphaeroides COX may induce a slowed proton uptake. Both of these effects will cause a decreased efficiency of electron transfer activity, and the difference in mechanism of inhibition between the two enzymes could be explained by variation in subunit structural homology between the two COX forms. Overall, it is clear that subunit III of cytochrome c oxidase, which lacks metal redox active centers, continues to reveal its significance to the catalytic mechanism and maintaining structural integrity of the enzyme.