The Hydroxylamine Oxidizing System of Nitrosomonas europaea

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

Hooper 0093447 The objectives of this research include understanding the catalytic mechanism of (a) The monoheme cytochrome P460, (b) the multiheme c-cytochrome, hydroxylamine oxidoreductase (HAO). (c) the tetraheme cytochrome c554, (d) the monoheme cytochrome c552 and (e) the di-c-heme cytcperoxidase. These are reactive cyochromes which participate in the energy-generating reactions of the autotrophic bacterium, N. europaea. The bacterium catalyzes a step in the nitrification pathway of the global nitrogen cycle. Its activity is key in agriculture, waste water treatment, on-site bioremediation of environmental pollutants, greenhouse gas formation and ozone depletion. The active site of the hydroxylamine oxidizing enzyme contains a novel heme-like macrocycle, heme P460, a derivative of a c-heme which has a unique, covalent crosslink to a protein tyrosine. A similar crosslink, but to a lysine, is observed in another hydroxylamine oxidizing enzyme from N. europaea called cytochrome P460. Both heme P460s have similar unique optical properties. . Catalytic hemes play critical roles in many cellular processes functioning as either reductases or oxygenases. HAO, on the other hand, catalyzes a biologically relevant dehydrogenation reaction, a function unprecedented among catalytic cytochromes, and rarely observed in metalloproteins in general. The mechanism to be tested includes: deprotonation of substrate; involvement of the cross-linked tyrosine or lysine in catalysis; and facilitation of electron removal by c-hemes. Specific goals are as follows. (a) Cytochrome P460. To purify the existing site directed mutant proteins K70A, K70R and K70Y and characterize their catalytic, optical-, EPR- and Raman-spectra and their redox potentials. To work on the X-ray crystallographic structure will continue. (b) HAO. To develop an expression system for the hao gene and make site-directed mutants. To identify by proteolysis and MALDI residue(s) which are derivatized by trinitromethane or which are modified in the native protein. To complete the X-ray structure. (c) Cytochrome c554. To characterize the existing site directed mutant F156A. (d) Cytochrome c552. To purify and characterize the existing v63 deletion site directed mutant and establish the structural basis for the HALS signal. (e) Cytochrome c Peroxidase. To complete analysis of the crystal structure and prepare site-directed mutants.