Crystal structure of the putidaredoxin reductase x putidaredoxin electron transfer complex.

In the camphor monooxygenase system from Pseudomonas putida, the [2Fe-2S]-containing putidaredoxin (Pdx) shuttles electrons between the NADH-dependent putidaredoxin reductase (Pdr) and cytochrome P450(cam). The mechanism of the Pdr.Pdx redox couple has been investigated by a variety of techniques.

Production and characterization of a functional putidaredoxin reductase-putidaredoxin covalent complex.

In the cytochrome P450cam-dependent monooxygenase system from Pseudomonas putida, putidaredoxin (Pdx) shuttles electrons between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with both partners. 1-Ethyl 3-[3-(dimethylamino)propyl]carbodiimide (EDC) was found to promote formation of stoichiometric Pdr-Pdx complexes only when carboxyl groups on Pdx were activated. The yield of the EDC-mediated cross-link depended on the Pdx variant used and the redox state of both partners, decreasing in the following order: Pdr(ox)-Pdx(ox) > Pdr(ox)-Pdx(red) > or = Pdr(red)-Pdx(red).

Mechanism of interaction between the general anesthetic halothane and a model ion channel protein, I: Structural investigations via X-ray reflectivity from Langmuir monolayers.

We previously reported the synthesis and structural characterization of a model membrane protein comprised of an amphiphilic 4-helix bundle peptide with a hydrophobic domain based on a synthetic ion channel and a hydrophilic domain with designed cavities for binding the general anesthetic halothane. In this work, we synthesized an improved version of this halothane-binding amphiphilic peptide with only a single cavity and an otherwise identical control peptide with no such cavity, and applied x-ray reflectivity to monolayers of these peptides to probe the distribution of halothane along the length of the core of the 4-helix bundle as a function of the concentration of halothane. At the moderate concentrations achieved in this study, approximately three molecules of halothane were found to be localized within a broad symmetric unimodal distribution centered about the designed cavity.

Redox-dependent changes in molecular properties of mitochondrial apoptosis-inducing factor.

Mitochondrial apoptosis-inducing factor (AIF) is a central player in the caspase-independent cell death pathway whose normal physiological function remains unclear. Our study showed that naturally folded mouse AIF very slowly reacts with NAD(P)H (k cat of 0.2-0.

Monolayers of a model anesthetic-binding membrane protein: formation, characterization, and halothane-binding affinity.

hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques.