The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.

Analyses. 1. VPC Analysis. Micellar or aqueous ketone-saturated photolysis mixtures were extracted with calibrated volumes of ethyl ether (containing an internal standard, typically hexadecane or benzophenone) and then subjected to quantitative VPC analysis. Product analyses were performed with a 3% OV-17/chrom-Q column (6 ft X in., column temperature 160-200 OC for DBK and as high as 240 OC for higher molecular weight photoinitiators). Products from scavenging by Cu2+ were also analyzed, relative to a standard, by using a lower column temperature (105-160 OC (10 OC/min) for detection of benzyl chlorides and benzyl alcohols). All products were identified on the basis of the known photochemistry of starting material as well as the known retention times of authentic compounds.Homogeneous photolysis mixtures composed of an organic solvent were typically treated with standard and then analyzed directly by VPC analysis (as described above).2. VPC/MS Analysis. For VPC/MS analysis of micellar photolysis mixtures, products were extracted with ether and the organic layer was submitted for analysis. A Finnigan 3300 with data system 6000 equipped with an OV-101 column and temperature programmed (150-200 "C) was employed using electron impact as the method of analysis.Data Treatment. Disappearance yields and product yields from photolyses were typically measured with respect to a standard. An unphotolyzed control sample was used to determine ketone disappearances as well as ensure proper mass balance of starting materials and products.Quantum yield or cage effect (by scavenging, see eq 2 and 6) determinations routinely involved preparation of 2-3 sets of samples per measurement. Only the averages of these analyses were used in the calculations. Acknowledgment. We thank the National Science Foundation and the Air Force Office of Scientific Research for their generous support of this research.Abstract: The magnetic circular dichroism (MCD) spectrum of chloro(meso-tetraphenylporphinato)iron(III) [ FeCI(TPP)] in dilute solution in polystyrene films has been measured over the temperature range 4-300 K. The MCD spectrum of this high-spin (S = 5 / 2 ) complex is dominated by paramagnetic effects ( C terms). The MCD temperature dependence at selected wavelengths is fit, assuming contributions from three ground-state Kramers doublets at energies 0, 20, and 60, where D is the quadratic axial zero-field splitting parameter. The 0 values obtained are independent of wavelength and in excellent agreement with previous measurements by other techniques. MCD is thus shown to be a usable technique for the determination of zero-field splitting parameters.

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.

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Nitrogenase (EC 1.18.6.1) catalyzes the conversion of dinitrogen to ammonia, the central reaction of biological nitrogen fixation. X-ray anomalous diffraction data were analyzed to probe the structures of the metal clusters bound by nitrogenase MoFe protein. In addition to one FeMo cofactor, each half-molecule of MoFe protein binds one large FeS cluster of a type not previously observed in a protein. The FeS cluster contains roughly eight Fe atoms, comprises two subclusters, and is separated from the FeMo cofactor by an edge-to-edge distance of 14 A. The inorganic framework of the FeMo cofactor is not resolved into subclusters, but the Mo atom is located at its periphery. FeMo cofactors in different half-molecules are 70 A apart and cannot promote binuclear activation of dinitrogen by two Mo atoms.