Spectral properties of bacterial nitric oxide reductase

Abstract

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.