Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.

Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme Fe B and high spin heme b 3 are -oxo bridged. The heme b 3 has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0 -8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b 3 remains oxidized. This results in a shift of the heme b 3 charge transfer band max to longer wavelengths. At pH 6.0 the charge transfer band max is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b 3 population is a mixture of both 605-and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b 3 in the three electronreduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.

Shewanella oneidensrsMR-1 is a Gram-negative facultative aerobe isolated from anaerobic frcshwater lake sediments. S. oneidensis MR-1 is capablc of respiratory-linked proton translocation and anaerobic growth coupled to the reduction of a wide range of terminal electron acceptors. When Shewanellais grown anaerobically the most abundant cytochrome produced is flavocytochrome c3. Pcc3 is the terminal clcctron acceptor in the anaerobic respiratory pathway t o fumarate. Scqucncing of the MR-I genome has led to the identification of four novel flavocytochromcs that share sequence identity to E'cc3. Molecular modelling studies of the novel flavocytochromc cnzymc active sites suggest thcsc enzymes to be monocarboxylatc rcductases. A wide range of acrylates, in particular phcnylpropanoids arc potential enzyme substrates. To determine the biological function, a gene-knockout of a Fcc enzyme has been constructcd. The null mutant will be grown with a variety of monocarboxylates as tcrminal electron acceptor. Over-expression and purification of a novel flavocytochrome for crystallisation studies is underway.Cobalamin is rcquircd as a coenzyme for at least two reactions in Pseudomonas aeruginosu, an opportunistic pathogen which is difficult to eradicate clinically due t o its multiple antibiotic rcsistance. In order to study the pathway, we have constructed two knockout mutants: one deficient in precorrin-4-mcthylase (cobM) and the other deficient in cobalt chelatase (cobN). The growth rates of thc wild type (wt) and the two knockout strains (cobMA and cobNA) were mcasurcd on minimal medium and, under aerobic conditions, were observed to be: wt > cobMA > cobNA. When grown in richer medium, the wild type produced approximately thrcc times more cobalamin under aerobic conditions than under anaerobic conditions. Ncithcr knockout mutant produced cobalamin under either of these conditions. Thus, the enzymes encoded by the cobM and cobN genes are required for both the acrobic and anaerobic production of cobalamin. However, cobalamin is not essential for the growth of I! aeruginosa under acrobic conditions although the organism can produce it in both the prcscncc and absence of oxygen.