Flavohemoglobin Hmp, but Not Its Individual Domains, Confers Protection from Respiratory Inhibition by Nitric Oxide in Escherichia coli

Abstract

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 M NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.

Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coli – Implications for urinary tract infection

“…Respiratory inhibition in response to NO is well established in non-pathogenic E. coli [3,6,43]. Respiration of UPEC was also significantly inhibited by NO and the inhibition was initiated momentarily upon NO addition and resumed after the NO levels decreased.…”

The flavohemoglobin BCFHG1 is the main NO detoxification system and confers protection against nitrosative conditions but is not a virulence factor in the fungal necrotroph Botrytis cinerea

“…cinerea has a single flavohemoglobin coding gene. Analysis of the deduced protein sequence and sequence homology studies indicate that it has the hemoglobin domain and the FADH and NADH binding domains that are characteristic of the flavohemoglobin proteins and essential for the NO degradation activity (Hernandez-Urzua et al, 2003). This structural similarity, together with the conservation in the B. cinerea BCFHG1 protein sequence of all the key residues thought to be important for nitric oxide dioxygenase function in bacterial flavohemoglobins (Frey and Kallio, 2003), residues which are also conserved in the sequences of functional flavohemoglobins from the yeasts S. cerevisiae and C. albicans (Ullmann et al, 2004), suggest that Bcfhg1 does indeed encode a functional flavohemoglobin involved in NO detoxification.…”

Thr-E11 Regulates O2 Affinity in Cerebratulus lacteus Mini-hemoglobin

“…These proteins include bacterial Hbs, flavohemoglobins, and invertebrate Hbs, with the most well studied examples being Vitreoscilla Hb (63-66), E. coli (HMP) flavohemoglobin (67)(68)(69)(70)(71)(72), and Ascaris Hb (73)(74)(75). The functions of these protein are still controversial and may involve O 2 sensing, NO dioxygenation, O 2 scavenging, and peroxidase activity.…”