title: Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae.
abstract: The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria--Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5 mM nitrite or at least 10 mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550 nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be -25 mV and -175 mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.
authors.affiliation: Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, Gent 9000, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Brigé
authors.forename: J A
authors.initials: JA
authors.lastname: Cole
authors.forename: W R
authors.initials: WR
authors.lastname: Hagen
authors.forename: Y
authors.initials: Y
authors.lastname: Guisez
authors.forename: J J
authors.initials: JJ
authors.lastname: Van Beeumen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006193:Haemophilus influenzae / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins