Related Experiment Video
Updated: Jun 17, 2026

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
Modulation of the active site conformation by site-directed mutagenesis in cytochrome c oxidase from Paracoccus
Hong Ji1, Tapan K Das, Anne Puustinen
1Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461, United States.
Abstract:
The structural and functional properties of active site mutants of cytochrome c oxidase from Paracoccus denitrificans (PdCcO) were investigated with resonance Raman spectroscopy. Based on the Fe-CO stretching modes and low frequency heme modes, two conformers (alpha- and beta-forms) were identified that are in equilibrium in the enzyme. The alpha-conformer, which is the dominant species in the wild-type enzyme, has a shorter heme a(3) iron-Cu(B) distance and a more distorted heme, as compared to the beta-conformer, which has a more relaxed and open distal pocket. In general, the mutations caused a decrease in the population of the alpha-conformer, which is concomitant with a decreased in the catalytic activity, indicating that the alpha-conformer is the active form of the enzyme. The data suggest that the native structure of the enzyme is in a delicate balance of intramolecular interactions. We present a model in which the mutations destabilize the alpha-conformer, with respect to the beta-conformer, and raise the activation barrier for the inter-conversion between the two conformers. The accessibility of the two conformers in the conformational space of CcO plausibly plays a critical role in coupling the redox reaction to proton translocation during the catalytic cycle of the enzyme.
Related Concept Videos
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Introduction to Mechanisms of Enzyme Catalysis
Ligand Binding and Linkage
Allosteric Regulation
Induced-fit Model
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical characteristics of...
Cooperative Allosteric Transitions

