Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alpha H195Q, alpha H195N, and alpha Q191 K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N-2 with the alpha H195Q and alpha H195N variants, but not with the alpha Q191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alpha H195Q or alpha H195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alpha Q191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze Fe-57 Mossbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alpha H195Q and alpha H195N MoFe proteins can bind N-2, but alpha Q195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N-2 reduction. (C) 2007 Elsevier Inc. All rights reserved.

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In this work (Al,Ti) and (Al,Ti,N) films with composition gradient in depth starting either with pure Al or pure Ti were deposited on Si, glass and Au at room temperature in a DC magnetron discharge without bias. The plasma parameters, for both custom made cathodes, were determined and the process was real-time controlled to obtain in the plasma the necessary deposition changes in relative metal abundances to get the desired depth profile composition on the films. In this work the process was designed to get a constant gradient for the composition depth profile. The morphology of the films was analysed by SEM while the composition gradients were measured by SIMS, XPS and RBS, confirming preset nominal depth composition profile of the films. To obtain (Al,Ti,N) thin films with gradient depth composition, N2 must be supplied to the discharges. The plasma behaviour is modified in the presence of N2 and the influence on the film characteristics is studied using the same techniques referred above. The (Al,Ti) and (Al,Ti,N) film properties are compared. We succeed in validating the coating technique opening new application possibilities.

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