Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N 2 O at the binuclear catalytic center consisting of high spin heme b 3 and non-heme Fe B . The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe B -NO and the penta-coordinated ferrous heme b 3 -NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time-and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N 2 O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the -oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR. Nitric-oxide reductase (NOR)1 is a metal-containing enzyme involved in biological denitrification processes. The enzyme catalyzes the two-electron reduction of nitric oxide (NO) to nitrous oxide (N 2 O) according to Equation 1 (1-4). Since the NO reduction reaction involves the N-O bond cleavage and the N-N bond formation, it is chemically interesting to elucidate the molecular mechanism of the NO reduction catalyzed by the metal enzymes.

We have developed the passive-type biofuel cell, in which two-electron oxidation of glucose and four-electron reduction of O2 occur at pH 7 in mediated bioelectrochemical processes, having the maximum power density of 5.0 mW/cm2 at 500 mV. This performance has been achieved by using a new electrolyte solution (Imidazole/HCl buffer, 2 M, pH 7), instead of the general solution (Sodium phosphate buffer, 1 M, pH 7); the maximum power density was a 3.0 mW/cm2 at the same conditions. By introducing the new electrolyte solution into our biofuel cell, we have succeeded in enhancing the catalytic current because the proton transfer into electrodes would increase compared with the phosphate buffer system.

The NH-pi interaction has been detected in the crystal structures of Co(III) ternary complexes with N,N-bis(carboxymethyl)-(S)-phenylalanine (BCMPA) and aromatic amino acids including (S)-phenylalanine ((S)-Phe), (R)-phenylalanine ((R)-Phe), and (S)-tryptophan ((S)-Trp)). Additionally, this interaction has been studied in solution for Co(III) ternary complexes with BCMPA or NTA (NTA = nitrilotriacetic acid) and several amino acids (AA) by means of electronic absorption, circular dichroism (CD), and (1)H NMR spectroscopies. The CD intensities of the Co(III) complexes with aromatic amino acids measured in the d-d region ( approximately 20.5 x 10(3) cm(-)(1)) are significantly decreased in ethanol solutions relative to water. Analogous complexes with aliphatic amino acids do not exhibit this solvent effect. The (1)H NMR spectra of the Co(III) complexes with aromatic amino acids measured in DMSO-d(6) exhibit upfield shifts of the NH peaks compared with those with aliphatic amino acids, which suggest a shielding effect due to the aromaticity. The upshift values coincide with those experimentally evaluated from the crystal structures. The magnitude of the upfield shifts agrees well with Hammett's rule, indicating that the increase of pi-electron densities on the aromatic rings leads attractive NH-pi interaction that exerts a larger shielding effect for the NH protons. In ligand-substitution reactions of the carbonatocobalt(III) complexes with amino acids, the yields of those with aromatic amino acids are higher than the yields obtained for complexes with aliphatic amino acids. This observation is discussed in connection with the important contribution of the NH-pi interaction as one of the promotion factors in the reaction.

The rhizobial FixL͞FixJ system, a paradigm of heme-based oxygen sensors, belongs to the ubiquitous two-component signal transduction system. Oxygen-free (deoxy) FixL is autophosphorylated at an invariant histidine residue by using ATP and catalyzes the concomitant phosphoryl transfer to FixJ, but oxygen binding to the FixL heme moiety inactivates the kinase activity. Here we demonstrate that ADP acts as an allosteric effector, reducing the oxygenbinding affinity of the sensor domain in FixL when it is produced from ATP in the kinase reaction. The addition of ADP to a solution of purified wild-type FixL resulted in an Ϸ4-to 5-fold decrease in oxygen-binding affinity in the presence of FixJ. In contrast, phosphorylation-deficient mutants, in which the well conserved ATPbinding catalytic site of the kinase domain is impaired, showed no such allosteric effect. This discovery casts light on the significance of homodimerization of two-component histidine kinases; ADP, generated in the phosphorylation reaction in one subunit of the homodimer, enhances the histidine kinase activity of the other, analogous to a two-cylinder reciprocating engine by reducing the ligand-binding affinity.