title: The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster.
abstract: Rieske and Rieske-type proteins are electron transport proteins involved in key biological processes such as respiration, photosynthesis, and detoxification. They have a [2Fe-2S] cluster ligated by two cysteines and two histidines. A series of mutations, L135E, L135R, L135A, and Y158F, of the Rieske protein from Thermus thermophilus has been produced which probe the effects of the neighboring residues, in the second sphere, on the dynamics of cluster reduction and the reactivity of the ligating histidines. These properties were probed using titrations and modifications with diethyl pyrocarbonate (DEPC) at various pH values monitored using UV-Visible and circular dichroism spectrophotometry. These results, along with results from EPR studies, provide information on ligating histidine modification and rate of reduction of each of the mutant proteins. L135R, L135A, and Y158F react with DEPC similarly to wild type, resulting in modified protein with a reduced [2Fe-2S] cluster in <90 min, whereas L135E requires >15 h under the same conditions. Thus, the negative charge slows down the rate of reduction and provides an explanation as to why negatively charged residues are rarely, if ever, found in the equivalent position of other Rieske and Rieske-type proteins.
authors.affiliation: Department of Chemistry, Trinity University, One Trinity Place, San Antonio, TX, 78212, USA.
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Karagas
authors.forename: Christie N
authors.initials: CN
authors.lastname: Jones
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Osborn
authors.forename: Anika L
authors.initials: AL
authors.lastname: Dzierlenga
authors.forename: Paul
authors.initials: P
authors.lastname: Oyala
authors.forename: Mary E
authors.initials: ME
authors.lastname: Konkle
authors.forename: Emily M
authors.initials: EM
authors.lastname: Whitney
authors.forename: R
authors.initials: R
authors.lastname: David Britt
authors.forename: Laura M
authors.initials: LM
authors.lastname: Hunsicker-Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017354:Point Mutation; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III; D004047:Diethyl Pyrocarbonate