Abstract
Spectroscopic and kinetic studies comparing the behavior of the recombinant cytochrome b reductase fragment of corn leaf nitrate reductase and a mutant in which cysteine 242 is replaced with a serine residue (C242S) have been carried out. The visible and circular dichroism spectra of the wild-type and mutant protein are virtually identical and compare well with those reported for nitrate reductases from other sources. The reduced wild-type protein forms a charge-transfer complex with NAD+ that has an absorption envelope that extends into the near infrared, with a maximum around 800 nm. The C242S mutant forms a similar charge-transfer complex with NAD+ but to a lesser extent than the wild-type. The reduction potential of the flavin for the wild-type protein is -287 mV, and that for the mutant is -279 mV. The rate of reduction by NADH of the C242S mutant is 7-fold slower than that for the wild-type protein, and the Kd is larger by a factor of 2. These results indicate that the cysteine 242 residue plays a role principally in facilitating electron transfer from NADH to the flavin rather than in binding of NADH to the enzyme.
Highlights
Nitrate reductase (EC 1.6.6.1–3) catalyzes pyridine nucleotide-dependent reduction of nitrate to nitrite as the first step in nitrogen assimilation in algae, fungi, and higher plants
It has been recognized that nitrate reductase has a cysteine that can be protected by pyridine nucleotides [1,2,3], and similar active site cysteines have been identified in other members of the ferredoxin NADPϩ reductase family of flavoenzymes [16, 19, 21, 23]
To determine if the invariant cysteine of the recombinant cytochrome b reductase fragment of nitrate reductase is the reactive one, each of the five cysteine residues of this fragment have been changed to another amino acid, and it is found that only by converting the cysteine 242 to a serine residue (C242S) is the ferricyanide reductase activity rendered insensitive to p-hydroxymercu
Summary
Nitrate reductase (EC 1.6.6.1–3) catalyzes pyridine nucleotide-dependent reduction of nitrate to nitrite as the first step in nitrogen assimilation in algae, fungi, and higher plants. The sequence similarity of the cytochrome b reductase fragment of nitrate reductase and ferredoxin NADPϩ reductase had been recognized before the structure was derived, but only a few key residues are strictly conserved [13, 14] Other members of this group include phthalate dioxygenase reductase, an FMN-containing NADH-dependent bacterial enzyme [15,16,17] and cytochrome b5 reductase [18]. The structural model for the cytochrome b reductase fragment has been extended to include an analysis of the C242S mutant, which indicates the side chain of the substituted serine forms a hydrogen bond with the main chain oxygen atom of glycine 147 that is not present in the wild-type protein This interaction causes a local conformational change that results in a large void in the active site [24]. We report a more detailed comparison of the spectroscopic and kinetic properties of the wild-type and C242S mutant of the recombinant cytochrome b reductase fragment in order to gain a better understanding of the role of cysteine 242 in flavin reduction by NADH
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