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Ferredoxin-dependent nitrate reductases (NR) play a role in the nitrate assimilation pathway of cyanobacteria. The cyanobacterial enzyme, which catalyzes two-electron reduction of nitrate into nitrite, is unique among nitrate reductases in using ferredoxin (Fd) as the sole physiological electron donor. NR from the cyanobacterium Synechococcus sp. PCC 7942 (NarB) is a 78 kDa, soluble, monomeric enzyme which contains a single [4Fe-4S] cluster and a single Mo bis-molybdopterin guanine dinucleotide (MoMGD) center as its only prosthetic groups. Kinetic studies, substrate-binding studies, computer modeling and amino acid replacements were used to investigate the details of the mechanism of the reaction catalyzed by this enzyme. Site-specific mutagenic replacements variants of four highly conserved basic NR amino acids (Lys58, Arg70, Lys130 and Arg146) were made and their properties investigated. Charge-replacement and charge-conserving variants of Lys58 and Arg70 were inactive with both the physiological electron donor, reduced Fd and with a non-physiological electron donor, reduced methyl viologen (MV). Replacement of Lys130 by glutamine produced a variant that showed a substantial loss in activity both with MV and Fd, while replacement by arginine produces a variant that retained a significantly higher activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. The roles of five amino acids, Cys148, Met149, Met306, Asp163, and Arg351, likely to be present at the active site of NR were analyzed using a site-directed mutagenesis approach. In addition to the differences in enzyme activity and substrate binding of the variants, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were also analyzed. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either reduced Fd, or reduced MV, as the source of electrons and all exhibited changes in either Mo content or the EPR properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The effects of these replacements on substrate binding were considerably smaller than was the case for the effects on the rates. The roles of three NR amino acids (Arg46, Lys614 and Arg43) predicted to be present at the NR/Fd docking interface by an in silico docking model calculated as part of this dissertation were also investigated by site-directed mutagenesis. Site-directed mutagenesis, coupled with activity measurements, metal analysis and EPR studies, implicated the side chains of Arg46, Lys614 and Arg43 as providing positive charges on nitrate reductase that are necessary for the productive binding of ferredoxin, but are not required for the assembly of functional Mo cofactor or [4Fe-4S] centers. In addition to these three positively charged NR amino acids present at the docking interface, the role of the seven negatively charged amino acids of Synechocystis sp PCC 6803 Fd, i.e., Glu29, Glu30, Asp60, Asp65, Asp66, Glu92 and Glu93, predicted to form complex with NR, was also investigated using site directed mutagenesis. These experiments identified four Fd amino acids, i.e., Glu29, Asp60, Glu92 and Glu93, are essential for the Fd binding and efficient electron transfer to the NR. Isothermal titration calorimetry (ITC) measurements showed that the most likely stoichiometry for the wild-type NR/wild-type Fd complex is 1:1, a Kd value 4.7 μM for the complex at low ionic strength residues. Both the enthalpic and entropic components associated with complex formation are favorable. ITC titrations of wild-type NR with four Fd variants, E29N, D60N, E92Q and E93D, demonstrated that the complex formation, although favorable, was less energetically favorable when compared to complex formation between the two wild-type proteins, suggesting that these negatively-charged Fd residues at these positions are important for the effective and productive interaction with wild type enzyme. The kinetics of electron transfer from reduced Fd to NR and to the nitrate was studied by flash-absorption spectroscopy. In the presence of nitrate, the rate of enzyme reduction shows a biphasic concentration dependence. At low enzyme concentrations (≤ 2 μM) the concentration dependence is approximately linear, with an estimated second-order rate constant of 7.4 ± 0.8 × 107 M 1s 1. At higher enzyme concentrations (>2 µM), the rate increases non-linearly to an asymptotic value of approximately 300 s 1. In presence of nitrate, the one-electron reduced enzyme shows essentially complete reduction of its Mo center with little or no contribution from reduction of the iron-sulfur cluster. The behavior of the enzyme in the absence of nitrate is significantly different from those obtained in the presence of this substrate. |
