Flavocytochrome <I>b</I><SUB>2</SUB> is a L-lactate dehydrogenase found in the intermembrane space of certain yeasts. Its physiological partner is cytochrome <I>c. </I> 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 <I>b</I><SUB>5</SUB>. The two domains are joined by an inter-domain hinge. A computer model of how a complex may be formed between flavocytochrome <I>b<SUB>2</SUB> </I>and cytochrome <I>c</I> 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 <I>b</I><SUB>2</SUB> were aligned with the positive arginines and lysines of cytochrome <I>c. </I>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 <I>b</I><SUB>2</SUB> 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 <I>c. </I>The model predicted a key residue for complex formation on flavocytochrome <I>b</I><SUB>2</SUB>, 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 <I>c</I> of 37.7 μM<SUP>-1</SUP>s<SUP>-1</SUP>, within the value for the wild-type enzyme of 34.8 μM<SUP>-1</SUP>s<SUP>-1</SUP> (pH 7.5, <I>I </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 <I>c</I> binds to flavocytochrome <I>b</I><SUB>2</SUB>. However, with the aid of molecular graphics and the sequence homology conserved within the 'cytochrome <I>b</I><SUB>5</SUB> fold', several residues were postulated to form a binding site for cytochrome <I>c. </I>
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661882 |
| Date | January 1998 |
| Creators | Short, Duncan M. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11915 |
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