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The diheme cytochrome c-554 which participates in ammonia oxidation in the chemoautotroph , Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c-554 at neutral pH is similar classical 6-coordinate low-spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c-554 suggests a 5-coordinate state which is unusual for c hemes. The oxidized spectrum closely resemble that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core-size vibrations indicates that both heme irons of c-554 have similar geometries.

When a total soluble extract of Nitrosomonas europaea was denatured with dodecyl sulphate, subjected to dodecyl sulphate/polyacrylamide-gel electrophoresis and illuminated with near-u.v. light, eight bands of protein fluorescence were observed. All but one of these bands were red in colour, a property characteristic of c-type cytochromes. Standard techniques were used to purify soluble c-type cytochromes from this organism, and it was then possible to assign all but two very minor bands to specific c-type cytochromes, namely hydroxylamine oxidase, cytochrome c-554, cytochrome c-552 and a cytochrome c-550 not previously described. The eight band had fluorescence peaking in the green region of the spectrum, probably caused by covalently bound flavin, and co-purified with hydroxylamine oxidase. The following physical properties were determined for these components: isoelectric point, molecular weights according to gel filtration and mobility on dodecyl sulphate/polyacrylamide gels, and alpha-band spectra at room temperature and 77K. Redox potentials were measured as follows: cytochrome c-554, E(m,7) = +20mV; cytochrome c-552, E(m,7) = +230mV; cytochrome c-550, E(m,7) = +140mV. When washed membranes were applied to dodecyl sulphate/polyacrylamide gels in the same way, a number of fluorescent bands were observed that could be matched by soluble proteins. In addition, there was one band that could not be detected in supernatants, migrating with an apparent molecular weight of 24000. This species is probably coincident with a c-type cytochrome having E(m,7) = +170mV found in redox titration of these membranes. In future studies, gel fluorescence should form a useful complement to spectroscopy for analysis of cytochrome composition in active cell-free preparations or semi-purified material.

After inoculation of a stationary-phase culture of Nitrosomonas europaea into fresh growth solution, the cell-associated orthophosphate increased rapidly to 800 mumoles/g (wet weight), whereas the acid-insoluble long-chain polyphosphate content decreased rapidly to 22 mumoles/g. As growth proceeded, the orthophosphate content decreased rapidly to a level of 15 mumoles/g and the long-chain polyphosphate content gradually increased to 60 to 90 mumoles/g. When the pH of a culture of Nitrosomonas decreased during growth below approximately 7.4, the rate of nitrite and polyphosphate synthesis increased and the ratio of change in protein to change in nitrite decreased. When the pH of the culture was maintained above 7.6 throughout growth, polyphosphate accumulation, an increased rate of nitrite and polyphosphate synthesis, and a decreased ratio of change in protein to change in nitrite were not observed. Cells of Nitrosomonas apparently accumulated polyphosphate when adenosine triphosphate generated during the oxidation of ammonia to nitrite was not efficiently used to promote an increase in cell mass. The rapid hydrolysis of polyphosphate after the transfer of stationary-phase cells into fresh growth solution was found to be triggered primarily by the higher pH of the fresh growth solution. The efflux of orthophosphate during culture growth was not associated with a decrease in the pH of the growth solution. Data on the chemical composition of Nitrosomonas are presented.

Fatty acids in the lipids of 19 marine and terrestrial nitrifying bacteria have been analyzed. Ammonia-oxidizing bacteria have a very simple acid composition; palmitic and palmitoleic acid account for 96 to 100% of the total acids. The fatty acids of nitrite-oxidizing bacteria cover a wider range, from C(14) to C(19), but from two to four acids still account for more than 80% of the total acids. Branched iso- and anteiso-acids are present in traces only in 2 of the 19 bacteria. The chemical and morphological similarity between blue-green algae and these bacteria is discussed.

Freezing and thawing of Nitrosomonas, followed by centrifugation of the homogenate at 3,000 x g, resulted in a fraction which appeared to consist of an intact membrane-envelope complex and contained approximately 50% of the cell protein and more than 90% of the ubiquinone and cytochrome A-type mammalian cytochrome c oxidase activity. The supernatant fraction, resulting from subsequent centrifugation of the extract at 100,000 x g, contained approximately 50% of the cell protein and more than 80% of the B- and C-type cytochrome and P-463 and the enzymes glutamate dehydrogenase; hydroxylamine dehydrogenase; nitrite synthetase; nitrite reductase; and 2,6-dichlorophenolindophenol-, p-phenylenediamine-, pyrogallol-, and hydroquinone-oxidase. Data on the concentration of electron transport components in Nitrosomonas are presented.