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. One of the exceptions is x-ray crystallography as the native partners associate weakly and resist co-crystallization. Here, we present the 2.6-A x-ray structure of a catalytically active complex between Pdr and Pdx C73S/C85S chemically cross-linked via the Lys(409Pdr)-Glu(72Pdx) pair. The 365 A(2) Pdr-Pdx interface is predominantly hydrophobic with one central Arg(310Pdr)-Asp(38Pdx) salt bridge, likely assisting docking and orienting the partners optimally for electron transfer, and a few peripheral hydrogen bonds. A predicted 12-A-long electron transfer route between FAD and [2Fe-2S] includes flavin flanking Trp(330Pdr) and the iron ligand Cys(39Pdx). The x-ray model agrees well with the experimental and theoretical results and suggests that the linked Pdx must undergo complex movements during turnover to accommodate P450(cam), which could limit the Pdx-to-P450(cam) electron transfer reaction.

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). The Pdr-Pdx C73S/C85S conjugate was purified and characterized. Compared to the equimolar mixture of intact Pdr and Pdx, the fusion protein was more efficient in electron transfer to cytochrome c and, in the presence of saturating levels of P450cam, more effectively supported camphor hydroxylation. On the basis of our results, we conclude that (i) the cross-linked complex is physiologically relevant and represents a suitable model for mechanistic studies, (ii) molecular recognition between Pdr and Pdx is redox-controlled and assisted by the Glu72(Pdx)-Lys409(Pdr) charge-charge interactions, and (iii) the high specificity of the Pdr-Pdx couple may be due to finely tuned interactions at the protein-protein interface resulting in only one strongly preferred docking orientation leading to efficient FAD-to-[2Fe-2S] electron transfer.

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. At the lowest concentration achieved, of approximately one molecule per bundle, the halothane distribution became narrower and more peaked due to a component of approximately 19A width centered about the designed cavity. At higher concentrations, approximately six to seven molecules were found to be uniformly distributed along the length of the bundle, corresponding to approximately one molecule per heptad. Monolayers of the control peptide showed only the latter behavior, namely a uniform distribution along the length of the bundle irrespective of the halothane concentration over this range. The results provide insight into the nature of such weak binding when the dissociation constant is in the mM regime, relevant for clinical applications of anesthesia. They also demonstrate the suitability of both the model system and the experimental technique for additional work on the mechanism of general anesthesia, some of it presented in the companion parts II and III under this title.

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 0.2-0.01 s(-1)) forming tight, dimeric, and air-stable FADH2-NAD(P) charge-transfer complexes ineffective in electron transfer. FAD reduction is accompanied by a conformational change involving AIF-specific N-terminal and regulatory 509-559 peptides and the active site His 453, and it affects susceptibility of AIF to calpain and AIF-DNA interaction, the two events critical for initiating caspase-independent apoptosis. Based on our results, we propose that formation of long lived complexes with NAD(P)H and redox reorganization may be functionally important and enable AIF to act as a redox-signaling molecule linking NAD(P)H-dependent metabolic pathways to apoptosis.

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. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The alpha-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide.

A model membrane protein for binding volatile anesthetics.

Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4-3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism.