Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O 2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d 1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd 1 nitrite reductase from P. aeruginosa.

In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-1. The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme. Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be positioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.

The reoxidation of substrate-reduced trimethylamine dehydrogenase by the artificial electron acceptor ferricenium hexafluorophosphate was studied by stopped-flow spectroscopy. The rate constants for the two sequential one-electron transfers from the reduced 4Fe-4S center to ferricenium ions were measured, the first (ka = 49 s-1) being about 7 times greater than the second (kb = 7.3 s-1) at 20 degrees C and neutral pH. The temperature dependence of the second electron transfer was studied over the range 10-40 degrees C, and the rate constant ranged from 5.7 to 19.2 s-1. Analysis of the temperature perturbation of kb by Marcus theory yielded values for the reorganizational energy of 1.95 eV and the electronic coupling matrix element of 0.26 cm-1. An electron tunneling pathway distance of 13 +/- 0.7 A was calculated which correlates with the shortest pathway measured from the 4Fe-4S center to the protein surface using the crystallographic coordinates of trimethylamine dehydrogenase. Tyr-442 is implicated in facilitating electron transfer from the enzyme to ferricenium ions. The data suggest a location for the docking site on the surface of trimethylamine dehydrogenase for the physiological electron acceptor (ETF).