Flavohemoglobin Hmp, but not its individual domains, confers protection from respiratory inhibition by nitric oxide in Escherichia coli.
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 microM 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.
Hernández-Urzúa
Elizabeth
E
Laboratorio de Microbiología y Genética Molecular, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, P. O. Box 70-228, Mexico City, 04510, Mexico.
Mills
Catherine E
CE
White
Gregory P
GP
Contreras-Zentella
Martha L
ML
Escamilla
Edgardo
E
Vasudevan
Subhash G
SG
Membrillo-Hernández
Jorge
J
Poole
Robert K
RK
D000595:Amino Acid Sequence; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D002474:Cell-Free System; D004093:Dihydropteridine Reductase / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D005838:Genotype; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000502:physiology*; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D010101:Oxygen Consumption / Q000187:drug effects*; D010446:Peptide Fragments / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D006420:Hemeproteins; D010446:Peptide Fragments; D009599:Nitroprusside; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases