The reaction of trimethylamine dehydrogenase (TMADH) with trimethylamine has been studied by rapid-scanning stopped-flow spectroscopy and steady-state kinetics. The covalently bound 6-S-cysteinylflavin mononucleotide (FMN) cofactor is initially reduced by substrate and exhibits a limiting first order rate constant of 230 s(-1) at pH 7.5 and 30 degrees C. One electron is then transferred intramolecularly from the reduced FMNH2 to the oxidized [4Fe-4S]2+ center. This reaction is biphasic, and the extent of the reaction which corresponds to the faster and slower rates is dependent upon the concentration of trimethylamine. The limiting first order rate constants are 160 and 4 s(-1). At low substrate concentrations, the faster rate is dominant, and at high substrate concentrations, the slower rate is dominant. These results are used to develop a model for the reductive half-reaction of TMADH in which two molecules of substrate bind to TMADH. One binds at the active site of oxidized TMADH and is converted to products. A second molecule binds but is not converted to products and influences the rate of intramolecular electron transfer. Analysis of the transient kinetic data yielded apparent dissociation constants for trimethylamine of 36 and 148 mu M, respectively, for binding to the catalytic and noncatalytic sites. Steady-state kinetic studies indicated substrate inhibition which was best described by a model in which binding of a second molecule of trimethylamine causes a 10-fold reduction in k(cat) from 11 to 1.1 s(-1). This suggests that, at high substrate concentrations, the rate of the intramolecular electron transfer reaction has become sufficiently slow to be at least partially rate-limiting for the steady-state reaction. These kinetic data are interpreted in the context of the known crystal structure of TMADH. The mechanistic implications regarding long range electron transfer and possible physiologic significance of these findings are discussed.

The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here Histidine kinases are the major signal transducer in bacteria, playing critical roles in adaptation to external stresses. EnvZ from Escherichia coli is one of the most extensively studied histidine kinases in terms of the functional and structural aspects (1). EnvZ is a transmembrane histidine kinase located in the inner membrane and functions as an osmosensor. It consists of a periplasmic receptor domain; two transmembrane domains, TM1 and TM2; and a cytoplasmic domain. The cytoplasmic domain has the bifunctional histidine kinase activity of EnvZ and can be further divided into three functional domains: the linker domain, domain A, and domain B. Domain A and domain B form the catalytic core of bifunctional histidine kinase EnvZ, having both kinase and phosphatase functions (2). Biochemical and structural studies of both domain A and domain B revealed that domain A contains the autophosphorylation site of a conserved His 243 and that it is a dimerization domain of EnvZ forming a four-helical bundle, whereas domain B contains an ATP-binding pocket (2-4). Thus, domain A and domain B are generally termed DHp and CA domains, respectively (5).The linker domain of EnvZ connects TM2 and the cytoplasmic domain. Although it is not directly involved in the enzymatic activities of EnvZ, it is known that the linker domain plays an important role in signal transduction. This is supported by the fact that a number of point mutations within the linker domain of EnvZ caused perturbation of the proper response against osmotic stresses (6 -9). The analysis of signal-transducing proteins from histidine kinases, adenylyl cyclases, methylaccepting chemotaxis proteins, and phosphatases revealed that like EnvZ these proteins also contain linker-like domains connecting their transmembrane domains to the cytoplasmic domains (10, 11). Importantly secondary structure prediction of these domains showed that they share a highly conserved helix-turn-helix fold even though their amino acid sequences have low homology. Therefore, these domains are called HAMP domains because this common motif was shared with histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases. Mutational studies of HAMP domain of BarA (12), Aer (13), or NarX (14) also demonstrated that the HAMP domains play a crucial role in their signal transduction, supporting the notion that the HAMP domains are required for the proper signal transduction of histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (10, 11).To elucidate the mechanism of the signal transduction, functional hybrid histidine kinases between EnvZ and chemosensor Tar have been constructed by fusing the receptor domain and HAMP domain of Tar to the cytoplasmic kinase domain of EnvZ (15). The binding of aspartate to the receptor of this hybrid histidine kinase, termed Taz, m...

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Within the enzyme trimethylamine dehydrogenase [TMADH], intramolecular electron transfer occurs between a fully reduced covalently bound 6-S-cysteinylflavin [FMN] cofactor, and an oxidized iron-sulfur [4Fe-4S]2+ center. When the enzyme is reduced by substrate trimethylamine, the kinetics of this intramolecular electron transfer [ET] reaction are biphasic, suggesting that ET occurs via two alternative processes [Falzon, L., & Davidson, V.L. (1996) Biochemistry 35, 2445-2452]. The formation of the FMN semiquinone was monitored by stopped-flow spectroscopy, and the two rate constants for the biphasic reaction were determined at temperatures ranging from 12 to 37 degrees C. Analysis of these rate constants by ET theory yielded values of 2.2 eV for the reorganizational energy [lambda] associated with each reaction and electronic coupling [H(AB)] of 5.9 and 47 cm-1 for the slower and faster ET reactions, respectively. The analysis also predicted average theoretical distance between the two redox centers of 12.3 A for the slower reaction and 8.1 A for the faster reaction. These predicted distances correlate well with the known crystal structure of TMADH and the most efficient pathways for ET that were predicted from the known structure using the Greenpath program. This analysis suggests that for each reaction the ET event is rate-limiting, but coupled to a highly unfavorable non-ET process, and that binding of a second molecule of substrate to reduced TMADH decreases the efficiency of the intramolecular ET.