Vitreoscilla Haemoglobin - Peroxidase Activity and Metabolic Aspects

Abstract: The function of non-vertebrate haemoglobins (Hbs) appears more diverse than that of vertebrate Hbs, possibly reflecting the various demands to be met among their hosts. Sharing structural features with other haem proteins/enzymes, Hbs display a qualitative overlap in activities with functions extending beyond oxygen transport/storage. When subjected to oxygen deficient conditions, the obligate aerobic bacterium Vitreoscilla synthesises a homodimeric haemoglobin, VHb, envisaged to sustain respiratory growth by facilitating oxygen-delivery to the respiratory apparatus. Heterologously expressed in various host organisms, VHb has shown to exert positive effects on growth and product formation. Furthermore, results from recent studies imply that VHb may be involved in alleviating oxidative/nitrosative stress. Despite being extensively studied, the biological function(s) of VHb remains elusive. This thesis describes two approaches aiming to gain further knowledge about the potential functions and biotechnological usefulness of VHb, including both its ability to exhibit peroxidase activity in vitro and the effect of VHb expression on growth and metabolism in aerated cultures of Lactococcus lactis and salt-stressed cultures of Escherichia coli. Our investigation reveals for the first time that VHb does exhibit peroxidase activity comparable to other peroxidases. In addition, studies of mutant VHbs and an engineered single-chain dimer, double VHb (dVHb), show that the catalytic activity can be modified by local alterations in the proximal site structure without perturbation of the globin fold. This property may be exploited in various biotechnological applications; the preferred VHb variant could be chosen depending on the conditions of interest. The in vivo studies of VHb in L. lactis describe the hitherto uninvestigated expression of a haemoglobin in a host organism lacking fully functioning respiration capacity. Our results show that in aerated cultures supplemented with haemin, L. lactis cells expressing biologically active VHb reach higher final cell densities than control cells. Expression of native VHb and an engineered single-chain dimer, double VHb (dVHb), in E. coli subjected to high concentrations of NaCl (up to 1.1 M) result in higher final cell densities for dVHb-cells than control cells lacking VHb for all conditions studied. Furthermore, our results imply that dVHb-cells show less signs of oxidative stress than control cells.