The first catalytic step of the light-driven enzyme protochlorophyllide oxidoreductase proceeds via a charge transfer complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (

Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase*S⃞

We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin (AdoCbl)- and pyridoxal l-phosphate (PLP)-dependent enzyme that catalyzes the 1,2-rearrangement of the terminal amino group of d-ornithine to generate (2R,4S)-2,4-diaminopentanoic acid. We show by stopped-flow absorbance studies that binding of the substrate d-ornithine or the substrate analogue d-2,4-diaminobutryic acid (DAB) induces rapid homolysis of the AdoCbl Co–C bond (781 s–1, d-ornithine; 513 s–1, DAB). However, only DAB results in the stable formation of a cob(II)alamin species. EPR spectra of DAB and [2,4,4-2H3]DAB bound to holo-ornithine 4,5-aminomutase suggests strong electronic coupling between cob(II)alamin and a radical form of the substrate analog. Loading of substrate/analogue onto PLP (i.e. formation of an external aldimine) is also rapid (532 s–1, d-ornithine; 488 s–1, DAB). In AdoCbl-depleted enzyme, formation of the external aldimine occurs over long time scales (∼50 s) and occurs in three resolvable kinetic phases, identifying four distinct spectral intermediates (termed A–D). We infer that these represent the internal aldimine (λmax 416 nm; A), two different unliganded PLP states of the enzyme (λmax at 409 nm; B and C), and the external aldimine (λmax 426 nm; D). An imine linkage with d-ornithine and DAB generates both tautomeric forms of the external aldimine, but with d-ornithine the equilibrium is shifted toward the ketoimine state. The influence of this equilibrium distribution of prototropic isomers in driving homolysis and stabilizing radical intermediate states is discussed. Our work provides the first detailed analysis of radical-based catalysis in this Class III AdoCbl-dependent enzyme