Protein-protein interaction of cytochrome b561 in chromaffin vesicle membranes studied by two-dimensional blue-native/sodium dodecyl sulfate gel electrophoresis and co-immunoprecipitation analysis

We analyzed a protein-protein interaction in solubilized chromaffin vesicles using two-dimensional electrophoresis (1st, Blue-Native PAGE; 2nd, Tricine-SDS-PAGE). Cytochrome b561 band, which was verified by immunoblotting, was observed in the two-dimensional gel with an apparent molecular weight of ~100~400kDa. On the other hand, purified cytochrome b561 showed a monomeric band (28 kDa) in Blue-Native PAGE. These results indicated that cytochrome b561 interacts with other proteins in the chromaffin vesicles to form a large protein complex(es). To clarify the nature of the interaction, we performed co-immunoprecipitation experiments, where the solubilized membrane proteins were treated with immunopurified anti- b561 IgG antibodies followed by sedimentation with protein-A-Sepharose. We found that there were no other proteins co-sedimented with cytochrome b561. Since the immunopurified anti- b561 IgG antibodies bound specifically to the C-terminal hydrophilic portion of cytochrome b561 protein, we concluded that such binding of the IgG antibodies to the C-terminal portion might cause an inhibition of protein-protein interaction with other proteins in the solubilized state

Direct electrochemical analyses of human cytochromes b5 with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials

<p>Abstract</p> <p>Background</p> <p>Cytochrome <it>b</it>₅performs central roles in various biological electron transfer reactions, where difference in the redox potential of two reactant proteins provides the driving force. Redox potentials of cytochromes <it>b</it>₅span a very wide range of ~400 mV, in which surface charge and hydrophobicity around the heme moiety are proposed to have crucial roles based on previous site-directed mutagenesis analyses.</p> <p>Methods</p> <p>Effects of mutations at conserved hydrophobic amino acid residues consisting of the heme pocket of cytochrome <it>b</it>₅were analyzed by EPR and electrochemical methods. Cyclic voltammetry of the heme-binding domain of human cytochrome <it>b</it>₅(HLMW<it>b</it>₅) and its site-directed mutants was conducted using a gold electrode pre-treated with β-mercarptopropionic acid by inclusion of positively-charged poly-L-lysine. On the other hand, static midpoint potentials were measured under a similar condition.</p> <p>Results</p> <p>Titration of HLMW<it>b</it>₅with poly-L-lysine indicated that half-wave potential up-shifted to -19.5 mV when the concentration reached to form a complex. On the other hand, midpoint potentials of -3.2 and +16.5 mV were obtained for HLMW<it>b</it>₅in the absence and presence of poly-L-lysine, respectively, by a spectroscopic electrochemical titration, suggesting that positive charges introduced by binding of poly-L-lysine around an exposed heme propionate resulted in a positive shift of the potential. Analyses on the five site-specific mutants showed a good correlation between the half-wave and the midpoint potentials, in which the former were 16~32 mV more negative than the latter, suggesting that both binding of poly-L-lysine and hydrophobicity around the heme moiety regulate the overall redox potentials.</p> <p>Conclusions</p> <p>Present study showed that simultaneous measurements of the midpoint and the half-wave potentials could be a good evaluating methodology for the analyses of static and dynamic redox properties of various hemoproteins including cytochrome <it>b</it>₅. The potentials might be modulated by a gross conformational change in the tertiary structure, by a slight change in the local structure, or by a change in the hydrophobicity around the heme moiety as found for the interaction with poly-L-lysine. Therefore, the system consisting of cytochrome <it>b</it>₅and its partner proteins or peptides might be a good paradigm for studying the biological electron transfer reactions.</p

The Nonstructural Proteins of Nipah Virus Play a Key Role in Pathogenicity in Experimentally Infected Animals

Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V−), rNiV(C−), and rNiV(W−), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V−) and rNiV(C−) were lower than the other recombinants. The rNiV(V−), rNiV(C−) and rNiV(W−) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V−) and rNiV(C−) but not the rNiV(W−) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo

Reaction Intermediates of Nitric Oxide Synthase from <i>Deinococcus radiodurans</i> as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe^II–O₂ Complex

Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate <i>N</i>-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from <i>Deinococcus radiodurans</i> was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 μs. Subsequently, ferrous heme reacted with O₂ to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 10⁸ M^–1 s^–1. In the tetrahydrofolate (H₄F)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 10³ s^–1. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H₄F, this step did not proceed, suggesting that H₄F was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s^–1. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s^–1). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates