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Vitamin B12, the ‘antipernicious anaemia factor’, is required for human and animal metabolism. It was discovered in the late 1940s and its unique corrin ligand was revealed approx. 10 years later by X-ray crystallography. The B12-coenzymes are cofactors in various important enzymatic reactions and are particularly relevant in the metabolism of anaerobic microorganisms. Microorganisms are the only natural sources of the B12-derivatives, whereas most spheres of life (except for the higher plants) depend on these cobalt corrinoids.

Chlorophyll metabolism is probably the most visible manifestation of life. In spite of this, chlorophyll catabolism has remained something of a mystery until about 10 years ago. At that time, the first non-green tetrapyrrolic chlorophyll breakdown products from higher plants were discovered, and the structure of the first one of them was elucidated by modern spectroscopic methods. In the meantime, the essential structural features of chlorophyll catabolites and some of the biochemistry of chlorophyll breakdown in higher plants have been uncovered, as outlined in this article.

Proteins of the cysteine-rich protein (CRP) family (CRP1, CRP2, and CRP3) are implicated in diverse processes linked to cellular differentiation and growth control. CRP proteins contain two LIM domains, each formed by two zinc-binding modules of the CCHC and CCCC type, respectively. The solution structure of the carboxyl-terminal LIM domain (LIM2) from recombinant quail CRP2 was determined by multidimensional homo- and heteronuclear magnetic resonance spectroscopy. The folding topology retains both independent zinc binding modules (CCHC and CCCC). Each module consists of two orthogonally arranged antiparallel beta-sheets, and the carboxyl-terminal CCCC module is terminated by an alpha-helix. 15N magnetic relaxation data indicate that the modules differ in terms of conformational flexibility. They pack together via a hydrophobic core region. In addition, Arg122 in the CCHC module and Glu155 in the CCCC module are linked by an intermodular hydrogen bond and/or salt bridge. These residues are absolutely conserved in the CRP family of LIM proteins, and their interaction might contribute to the relative orientation of the two zinc-binding modules in CRP LIM2 domains. The global fold of quail CRP2 LIM2 is very similar to that of the carboxyl-terminal LIM domain of the related but functionally distinct CRP family member CRP1, analyzed recently. The carboxyl-terminal CCCC module is structurally related to the DNA-binding domain of the erythroid transcription factor GATA-1. In the two zinc-binding modules of quail CRP2 LIM2, flexible loop regions made up of conserved amino acid residues are located on the same side of the LIM2 domain and may cooperate in macromolecular recognition.

Chlorophyll breakdown in green plants is a long-standing biological enigma. Recent work has shown that pheophorbide a (Pheide a) derived from chlorophyll (Chl) is converted oxygenolytically into a primary fluorescent catabolite (pFCC-1) via a red Chl catabolite (RCC) intermediate. RCC, the product of the ring cleavage reaction catalyzed by Pheide a oxygenase, which is suggested to be the key enzyme in Chl breakdown in green plants, is converted into pFCC-1 by a reductase. In the present study, an in vitro assay comprising 18O2 Pheide a oxygenase and RCC reductase yielded labeled pFCC-1. Fast atom bombardment-mass spectrometric analysis of the purified pFCC-1 product revealed that only one of the two oxygen atoms newly introduced into Pheide a in the course of the cleavage reaction is derived from molecular oxygen. Analysis of the fragment ions located the oxygen atom derived from molecular oxygen on the formyl group of pyrrole B. This finding demonstrates that the cleavage of Pheide a in vascular plants is catalyzed by a monooxygenase. Chlorophyll breakdown is therefore indicated to be mechanistically related in higher plants and in the green alga Chlorella protothecoides.

Members of the cysteine- and glycine-rich protein family (CRP1, CRP2, and CRP3) contain two zinc-binding LIM domains, LIM1 (amino-terminal) and LIM2 (carboxyl-terminal), and are implicated in diverse cellular processes linked to differentiation, growth control, and pathogenesis. Here we report the solution structure of full-length recombinant quail CRP2 as determined by multi-dimensional triple-resonance NMR spectroscopy. The structural analysis revealed that the global fold of the two LIM domains in the context of the full-length protein is identical to the recently determined solution structures of the isolated individual LIM domains of quail CRP2. There is no preference in relative spatial orientation of the two domains. This supports the view that the two LIM domains are independent structural and presumably functional modules of CRP proteins. This is also reflected by the dynamic properties of CRP2 probed by 15N relaxation values (T1, T2, and nuclear Overhauser effect). A model-free analysis revealed local variations in mobility along the backbone of the two LIM domains in the native protein, similar to those observed for the isolated domains. Interestingly, fast and slow motions observed in the 58-amino acid linker region between the two LIM domains endow extensive motional freedom to CRP2. The dynamic analysis indicates independent backbone mobility of the two LIM domains and rules out correlated LIM domain motion in full-length CRP2. The finding that the LIM domains in a protein encompassing multiple LIM motifs are structurally and dynamically independent from each other supports the notion that these proteins may function as adaptor molecules arranging two or more protein constituents into a macromolecular complex.