Use this URL to cite or link to this record in EThOS:
Title: The location of the cytochrome c binding site on flavocytochrome b2
Author: Short, Duncan M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Flavocytochrome b2 is a L-lactate dehydrogenase found in the intermembrane space of certain yeasts. Its physiological partner is cytochrome c. The enzyme exists as a homotetramer with each subunit consisting of two domains, a flavodehydrogenase domain and a cytochrome domain. The cytochrome domain is homologous with the extensively studied cytochrome b5. The two domains are joined by an inter-domain hinge. A computer model of how a complex may be formed between flavocytochrome b2 and cytochrome c was produced in 1993, and predicted several residues to be important for molecular recognition. In accordance with other simulated models of protein complexes, the negative aspartates and glutamates of flavocytochrome b2 were aligned with the positive arginines and lysines of cytochrome c. The haems were found to be parallel, with an iron-iron separation of 25.6Å. An electron-transfer pathway was also proposed between the two haems. This involved the side-chain of isoleucine 50, which is in contact with the flavocytochrome b2 haem, the backbone of lysine 51, and the aromatic ring of phenylalanine 52, which was reported to be in van der Waal's contact with the haem of cytochrome c. The model predicted a key residue for complex formation on flavocytochrome b2, glutamate 91. The construction of a mutant-enzyme, with glutamate 91 mutated to a lysine, produced a second-order rate constant for the reduction of cytochrome c of 37.7 μM-1s-1, within the value for the wild-type enzyme of 34.8 μM-1s-1 (pH 7.5, I = 0.1M, 25°C). The haem redox potential for the mutant-enzyme was -13mV, a value again within error of the wild-type enzyme, 017mV. These data, along with data from two other mutant-enzymes, showed that the hypothetical complex was not a realistic model of how cytochrome c binds to flavocytochrome b2. However, with the aid of molecular graphics and the sequence homology conserved within the 'cytochrome b5 fold', several residues were postulated to form a binding site for cytochrome c.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available