The two cytochromes c in the facultative methylotroph Pseudomonas AM1

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Abstract
It was previously suggested that there is only 1 soluble cytochrome c in Pseudomonas AM1, having a MW of 20,000, a redox midpoint potential of about +260mV and a low isoelectric point. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 now revealed the presence of 2 different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isolelectric focusing. MW were determined by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behavior of the 2 cytochromes on ion-exchange celluloses. The more abundant cytochrome CH (.lambda.max. 550.5 nm) had a low MW (11,000), a midpoint potential of about +294 mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mM-TrisHCl buffer, pH 8.0. The less abundant cytochrome CL (.lambda.max. 549 nm) was about 30% of the total; it had a high MW (20,900), a midpoint potential of about +256 mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20 mM-Tris/HCl buffer, pH 8.0. The pH-dependence of the midpoint redox potentials of the 2 cytochromes c were very similar. There were 4 ionizations affecting the redox potentials in the pH range studied (pH 4.0-9.5), 2 in the oxidized form (pK values about 3.5 and 5.5) and 2 in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the 2 propionate side chains of the heme. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C1 compounds, although still able to grow well on multicarbon compounds such as succinate. Wether or not these 2 cytochromes c have separate physiological functions is not yet certain.