The key metalloprotein of dissimilator nitrite reductase found in the majority of microbial denitrifiers is called cytochrome cd which contains an extractable green heme prosthetic group. This so-called -d-l heme has been assumed to be a chlorin since the late 1950's. Recently a structure determination of isolated heme -d-l by another laboratory also favored a chlorin macrocycle. However, critical analyses of the available spectra data have led us to propose that the -d-1 heme is not a chlorin but a dioxoisobacteriochlorin. Recent experimental studies on demetalated heme -d-1 and model compounds have yielded convincing evidence to support the novel dioxo ring structure. The objectives of the proposed research are (1) to establish unambiguously the structure of the -d-1 heme; (2) to elucidate the intrinsic physicochemical properties of this ring and to identify the attributes which make this heme uniquely suitable for mediating the nitrite reduction; and (3) to understand the reaction pathways and mechanisms. Our experimental approach includes (1) chemical derivatizations of isolated -d-1 for further confirmation of the proposed structure; (2) study of new synthetic methods to prepare dioxoisobacteriochlorins as models and analogues for -d-1 and ultimately, the total synthesis of -d-1; (3) systematic study and documentation of the spectroscopic properties and ligand coordination chemistry of heme -d-1 and model compounds to provide a knowledge base for this class of heme groups; (4) the substitution of synthetic metal dioxo-isobacteriochlorin complexes into the active sites of myoglobin and cytochrome cd to probe the structural basis of prosthetic group/protein interactions; and (5) tests of possible reduction pathways for dioxygen and nitrite reductions using heme models and reconstituted proteins. Denitrification is a basic energy coupled process of metabolism in a large variety of bacteria, including some pathogens. Furthermore, products of denitrification are NO-2-.NO and N20, all of which have direct or indirect toxicity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036520-02
Application #
3290638
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1986-04-01
Project End
1989-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
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Andersson, L A; Loehr, T M; Wu, W S et al. (1990) Modelling heme d1. The spectral properties of copper(II) porphyrindiones. FEBS Lett 267:285-8
Salehi, A; Oertling, W A; Fonda, H N et al. (1988) Resonance Raman spectra of the II-cation radicals of copper, cobalt, and nickel methyloctaethylchlorins: vibrational characteristics of chlorophyll models. Photochem Photobiol 48:525-30
Chang, C K; Timkovich, R; Wu, W (1986) Evidence that heme d1 is a 1,3-porphyrindione. Biochemistry 25:8447-53