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J. Biol. Chem., Vol. 278, Issue 37, 34975-34982, September 12, 2003

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

Elizabeth Hernández-Urzúa  , Catherine E. Mills  ¶, Gregory P. White ||, Martha L. Contreras-Zentella  **, Edgardo Escamilla **, Subhash G. Vasudevan || , Jorge Membrillo-Hernández  and Robert K. Poole ¶ 

From the 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, ^¶Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom, ^||Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4811, Australia, and ^**Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, P. O. Box 70-242, Mexico City 04510, Mexico

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 µM 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.

Received for publication, April 8, 2003 , and in revised form, June 13, 2003.

^* This work was supported by Biotechnology and Biological Sciences Research Council Grants PRS12199 and P18939 (to R. K. P.), Consejo Nacional de Ciencia y Tecnologia (CONACyT) México Grant J-33369 and Progama de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT)-Universidad Nacional Autónoma de México Grant IN5200 (to J. M.-H.), and a Ph.D. CONACyT studentship (to E.H-U.). The Royal Society, the Wellcome Trust, and Academia Mexicana de Ciencias generously provided traveling support to J. M.-H. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

Present address: Novartis Institute for Tropical Diseases, 1 Science Park Rd., Singapore 117528.

To whom all correspondence should be addressed. Tel.: 44-114-222-4447; Fax: 44-114-272-8697; E-mail: r.poole{at}sheffield.ac.uk.

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