Probing the unusual oxidation/reduction behavior of Paracoccus pantotrophus cytochrome cd1 nitrite reductase by replacing a switchable methionine heme iron ligand with histidine.
A M106H variant, where M106 is a c-type heme iron axial ligand, of cytochrome cd(1) nitrite reductase is an inactive protein in vivo. Expression of the holoprotein in Paracoccus pantotrophus required generation of nitric oxide during cell growth through simultaneous expression of an exogenous copper nitrite reductase from Alcaligenes faecalis. In the absence of the latter protein, only a semi-apo form of M106H cytochrome cd(1) was formed. Thus it was demonstrated that expression of the chromosomal nir genes for d(1) heme biosynthesis in P. pantotrophus is NO-dependent, probably mediated by the transcription factor NNR, and a route to low or zero activity mutants had been established. The value of such variants for mechanistic studies on cytochrome cd(1) is illustrated by the use of M106H to demonstrate that the d(1) heme potential can be resolved and measured at approximately +175 mV with the c heme shifted to -60 mV, consistent with its bishistidinyl coordination. The unusual highly cooperative and strongly hysteretic redox titration of the wild type is lost in the M106H protein. The same c heme midpoint potential was observed in a M106H variant of a c-domain construct. The difference between d(1) heme and c heme redox potentials has allowed preparation of a M106H protein with oxidized c heme and reduced d(1) heme. This one electron reduced form will reduce nitrite to nitric oxide, but the latter remains bound to the resulting fully oxidized enzyme.
Zajicek
Richard S
RS
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Cartron
Michaël L
ML
Ferguson
Stuart J
SJ
D000595:Amino Acid Sequence; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D008715:Methionine / Q000378:metabolism*; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D042084:Paracoccus pantotrophus / Q000201:enzymology*
D003580:Cytochromes; D008024:Ligands; D006418:Heme; D006639:Histidine; D008715:Methionine; D007501:Iron; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming)