Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of −310 mV via Mia40 with an intermediate midpoint potential of −290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of −150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX9C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.

Two new methods for simultaneous measurement of velocity and internal state are reported, and results are presented for application of the techniques to the photodissociation of CH3I. The internal state of the probed fragment is chosen by tuning the resonant ionizing laser, while the fragment velocity is determined from the arrival time distribution of fragment ions at the detector of a time-of-flight (TOP) mass spectrometer. For the 266-nm dissociation of CH3I or CD3I, the amount of I*(1 2Pi/2) vs I(2P3/2) produced in coincidence with CH3(v2=i) or CD3(v2=y) has been determined for i = 0-2 and j = 0-3.The values are (i, I/I*) = (0, 0.08), (1, 0.37), (2, 1.1) and (j, I/I*) = (0, <0.05), (1, 0.09), (2, 0.19), (3, 0.68). These I/I* ratios were found to vary dramatically with probe wavelength, partly due to variations in ratio with methyl rotational level and partly due to different contributions from overlapping vibronic bands, in a further application, separate MPI wavelength scans were obtained simultaneously for CD3 produced in coincidence with I and I*. Observations on the I and I* fragments have allowed us to determine values for the anisotropy parameters (/3(I) = 1.7 ± 0.1, /3(I*) = 1.8 ± 0.1). Application of these techniques to the detection of clusters and to the discrimination between multiple pathways to the same fragment ion in multiphoton dissociation and ionization is discussed.to the Doppler method but requires neither sub-Doppler laser line (1)

The photodissociation dynamics of CH)I and CD31 have been examined by using multiphoton ionization to probe the CH), CD 3 , I( =5 2P3/2) and 1*( =15 2P 1/2 ) photoproducts. The parent compounds were cooled in a supersonic expansion, collimated into a molecular beam, and dissociated at 266 nm. For the CD31 dissociation, the ratio ofCD 3 (v = O)/(v = 2) was estimated to be about 1.1, with mUltiple determinations ranging from 0.47-2.1. The quantum number v here denotes the nascent excitation of the V 2 "umbrella" mode. Measurements of the CD 3 (v = 1) and (v = 3) vibronic bands indicated that the (v = 1) I (v = 3) ratio is greater than unity, with some measurements suggesting values as large as 10. A value for the CH 3 (v = O)/(v = 2) ratio from dissociation ofCH 3 1 could not be estimated, although it was clearly larger than that for CD 3 . The CH 3 (v = 0) and CD 3 (v = 0) products from this dissociation are fit by 120 ± 30 K and 105 ± 30 K rotational distributions, respectively. The dissociation mechanism produces alignment in the molecular frame such that there is a strong preference for K = 0 (rotation perpendicular to the top axis). Assuming that the relative velocity vector lies along the CH 3 C 3 axis, the velocity and rotation vectors tend to be perpendicular. It is likely that K = 0 molecular frame alignment is produced in photodissociation through both the I and 1* channels.ciation showed maximum popUlations of CH 3 in v = 2 and 4 4222

The photodissociations of OCS at 157 nm and of CH31(CD,I) at 266 nm have been investigated by using tunable vacuum ultraviolet laser-induced fluorescence and multiphoton ionization to probe the CO or S and the CH,(CD,) or I photoproducts, respectively. In the OCS dissociation, sulphur is produced almost entirely in the S('S) state, while CO is produced in its ground electronic state and in vibrational levels u = 0-3 in the approximate ratio3). The rotational distribution for each vibrational level is found to be near-Boltzmann, with temperatures that decrease from 1350 K for u = 0 to 770 K for v = 3. Measurements of the CO Doppler profiles demonstrate that the dissociation takes place from a transition of predominantly parallel character ( p > 1.3) and that the CO velocity and angular momentum vectors are perpendicular to one another. In the CD31 dissociation, the ratio of CD3 ( u = O)/( u = 2) was estimated to be ca. 1.1, with multiple determinations in the range 0.47-2.1. The quantum number u here denotes the nascent excitation of the u2 'umbrella' mode. A value for the CH, ( u = O)/( u = 2) ratio from dissociation of CH31 could not be estimated, although it was clearly larger than that for CD,. The CH3 ( u = 0) and CD, ( u = 0) products from this dissociation are fitted by 120* 30 K and 105 f 30 K rotational distributions, respectively. The dissociation mechanism produces alignment in the molecular frame such that there is a strong preference for low values of K . Assuming that the relative velocity vector lies along the CH, C3 axis, then the velocity and rotation vectors tend to be perpendicular.

Multiphoton ionization spectra have been obtained and analyzed for excitation in the 215–360 nm region from the X 3Σ−g, a 1Δg, and b 1Σ+g states of O2. The 0–0 band of the C 1Πg state is reported for the first time. Measurements of other vibrational bands terminating in the C 3Πg and d 1Πg states are in good agreement with determinations by other groups. Several vibrational levels (v′=0–5) of the 3dπg Rydberg complex have been assigned on the basis of (1) an analysis of the spin–orbit couplings between the (Λ,S) basis-set states, (2) spectral simulation, and (3) the behavior of the states when the excitation radiation is changed from linear to circular polarization.

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