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.

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 Pdr⅐Pdx 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-Å x-ray structure of a catalytically active complex between Pdr and Pdx C73S/C85S chemically cross-linked via the Lys 409Pdr -Glu 72Pdx pair. In Pseudomonas putida, the camphor monooxygenase system uses NADH as a source of electrons and consists of three soluble proteins: FAD-containing putidaredoxin reductase (Pdr, 3 45.6 kDa), [2Fe-2S]-containing putidaredoxin (Pdx, 11.4 kDa), and cytochrome P450 cam (P450 cam , 46.6 kDa) (1). Pdx receives reducing equivalents from Pdr in two one-electron steps and delivers them one at a time to P450 cam . Acting as a shuttle, Pdx forms transient electron transfer (ET) complexes with its redox partners during turnover (2-6). The x-ray structures of all components of the camphor monooxygenase have been determined (7-10), but neither Pdr⅐Pdx nor Pdx⅐P450 cam native complexes have been crystallized thus far.The mechanism of interaction and interprotein ET in the Pdr⅐Pdx pair has been the focus of several research groups including ours. Significant progress has been made in the general understanding of how the Pdr⅐Pdx complex is formed and functions. In particular, both two-and one-electron reduced species of Pdr were identified as catalytically competent redox intermediates (3, 4, 11); ionic and hydrophobic interactions as well as steric complementarity were proven to contribute to molecular recognition between the partners (4, 12, 13) and involve Tyr 33 , Asp 38 , Arg 66 , Glu 72 , and Cys 73 of Pdx (14 -16); and, based on the x-ray structures of the flavo-and iron-sulfur proteins (8, 10, 17), a computer model for the Pdr⅐Pdx ET complex was generated and experimentally tested (16). However, the precise manner of the Pdr-Pdx interaction, the docking sites, and interface residues remained unknown. The most direct way to obtain this information would be determination of the x-ray structure of the Pdr⅐Pdx complex, but, despite our multiple attempts, the native proteins resisted co-crystallization. This could in part be due to weak association (K d ϭ 66 M) (16) and quick inactivation of wild type Pdx in solution (8).To overcome this problem, we attempted to produce a functionally active Pdr⅐Pdx conjugate. Having screened various cross-linking agents, reaction conditions, and Pdx mutants, we prepared a catalytically competent complex between wild type Pdr and Pdx C73S/C85S covalently linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) via the Lys 409Pdr -Glu 72Pdx salt bridge (6). The double mutant of Pdx was chosen for a functional analysis because substitution of cysteines 73 and 85 with serine, a highly isosteric analogue, only mildly aff...

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.

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.

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.