The two-haem nitrite reductase of Micrococcus denitrificans.

Abstract 1. 1. This work characterises a two-haem cytochrome isolated from Micrococcus denitrificans . The ratio of the absorbance of the protein band at 280 nm to that of the Soret band at 408 nm of the purest preparation was 0.75, the molecular weight was 120 000 and the isoelectric point was pH 3.85(0°). In addition to the major component I, there are two slightly less acidic minor components. Component I and Components II + III have been separated and compared spectroscopically. 2. 2. The two-haem cytochrome contains a c- type and an a 2 ( d ) like haem. The c- haem must be bound to the protein in an unusual manner, being repsonsible for the double α-band and the low α/β band ratio in the reduced native cytochrome; in alkaline pyridine a normal c- type haemochrome is formed, with a single sharp band at 550 nm. The d- like (green) haem reacts under different conditions with CO 2 pyridine, imidazole, CN-, N 3 - and NO 2 -; it can be removed from the protein by mild procedures and is not identical with haem d . 3. 3. This cytochrome is present only in cells grown anaerobically in the presence of NO 3 - and it has both nitrite reductase and cytochrome c oxidase activities when assayed with reduced Micrococcus cytochrome c as substrate. 4. 4. Pseudomonas aeruginosa cytochrome oxidase/nitrite reductase was prepared for comparison ( A 280 nm /A 410 nm −0.80 ). It is a less acidic (isoelectric point 7.1) and smaller (mol. wt. 85 000) protein, but contains the same two haem groups as the Micrococcus enzyme. Unlike the Micrococcus enzyme, it has only one component.