title: Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV-VIS absorption study.
abstract: Putidaredoxin (Pdx) is an 11,400-Da iron-sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV-VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3+/-0.5 degrees C and 28.4+/-0.5 degrees C. The 16.3 degrees C transition is attributed to the disruption of the hydrogen bonding of the active center bridging sulfur atom with cysteine 45 and alanine 46. The transition at 28.4 degrees C occurs exclusively in the Pdx(ox) at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx(red) and Pdx(ox) to a comparable extent, and their concentration ratio remains constant. In contrast, the 28.4 degrees C transition preferentially destabilizes Pdx(ox) thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding of the iron ligating cysteines 45, 39 with residues threonine 47, serine 44, glycine 41, and serine 42 causes the 28.4 degrees C transition. The sensitivity of the UV-VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.
authors.affiliation: Biotechnology Division, National Institute of Standards and Technology, Stop 8312, Gaithersburg, MD 20899-8312, USA. vytas@nist.gov
authors.forename: Vytas
authors.initials: V
authors.lastname: Reipa
authors.forename: Marcia
authors.initials: M
authors.lastname: Holden
authors.forename: Martin P
authors.initials: MP
authors.lastname: Mayhew
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Vilker
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry*; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: D005288:Ferredoxins; C100137:putidaredoxin; D013455:Sulfur; D007501:Iron; D003545:Cysteine; D000409:Alanine