Reduction by NADPH

It has been believed that P-450 reduction by NADPH cytochrome P-450 reductase is a biphasic process, but it was recently shown [7] that some P-450 cytochromes are reduced with single-exponential kinetics and that the presence of substrate is not an obligatory condition for the reduction of all P-450 forms. Thus, the kinetics of reduction of various ferric P-450 cytochromes possibly depends on many factors such as substrate, rate-limiting step, etc. 

Sulfite reductase catalyzes the six-electron reduction by NADPH of sol" to and NO2 to NH3. In E. coli this enzyme is a complex structure with subunit composition 0 8)84 (Siegel et al, 1982). The enzyme active site is on the /3 subunit, which contains both a 4Fe 4S cluster and a siroheme prophyrin. Substrates and ligands have been found to bind to the siroheme. The a subunit binds NADPH and serves to shuttle electrons to the active site through bound FAD and FMN groups. Isolated )8 subunits can catalyze sulfite reduction in the presence of a suitable electron donor. 

Peterson, J. A., and Boddupalli, S. S. (1992). P450BM-3 Reduction by NADPH and sodium dithionite. Arch. Biochem. Biophys. 294, 654-661. 

The pathway The first committed step in fatty acid biosynthesis is the carboxylation of acetyl CoA to form malonyl CoA which is catalyzed by the biotin-containing enzyme acetyl CoA carboxylase. Acetyl CoA and malonyl CoA are then converted into their ACP derivatives. The elongation cycle in fatty acid synthesis involves four reactions condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP releasing free ACP and C02, then reduction by NADPH to form D-3-hydroxybutyryl-ACP, followed by dehydration to crotonyl-ACP, and finally reduction by NADPH to form butyryl-ACP. Further rounds of elongation add more two-carbon units from malonyl-ACP on to the growing hydrocarbon chain, until the C16 palmitate is formed. Further elongation of fatty acids takes place on the cytosolic surface of the smooth endoplasmic reticulum (SER). 

Reduction by NADPH to form the P-hydroxy acid derivative ... 

Reduction by NADPH to make the saturated fatty acid ... 

Yoshimoto and Sato (4 1) have isolated sulfite reductases from four mutants of sulfite reduction by NADPH, but could utilize MVH as electron donor. All the mutant enzymes contained the chromophore responsible for the 386- and 587-nm peaks, nonheme iron, and labile sulfide. Three of these mutant enzymes contained FMN no flavin was detected in the fourth. The sedimentation coefficients of these preparations... 

These conclusions regarding the yeast sulfite reductase are essentially in agreement with our current knowledge of the mechanism of sulfite reduction by NADPH and MVH in the E. coli enzyme. 

Binding of 4-hydroxybenzoate (S) in the phenolate form facilitates flavin reduction by NADPH. After NADP+ release, the flavin hydroquinone reacts with molecular oxygen to yield the flavin C4a-hydroperoxide oxygenation species. Protonation of the distal oxygen of the peroxflavin facilitates the electrophilic attack on the nucleophilic carbon center of the substrate phenolate. After monooxygenation, the resulting hydroxyflavin is... 

Figure 26.2. Synthesis of an Ether Phospholipid. Steps in the synthesis include (1) acylation of dihydroxyacetone phosphate by acyl CoA, (2) exchange of an alcohol for the carboxylic acid, (3) reduction by NADPH, (4) acylation by a second acyl CoA, (5) hydrolysis of the phosphate ester, and (6) transfer of a phosphocholine moiety.

The biopterin hydroperoxide donates an electrophilic oxygen atom to the benzene ring, rather in the way of m-CPBA, though that reagent would not do the trick. The enzyme is essential. The other product is an oxidized biopterin and is recycled by reduction by NADPH back to the biopterin. 

Terpenoids do not necessarily contain exact multiples of five carbons and allowance has to be made for the loss or addition of one or more fragments and possible molecular rearrangements during biosynthesis. In reality the terpenoids are biosynthesized from acetate units derived from the primary metabolism of fatty acids, carbohydrates and some amino acids (see Fig. 2.10). Acetate has been shown to be the sole primary precursor of the terpenoid cholesterol. The major route for terpenoid biosynthesis, the mevalonate pathway, is summarized in Fig. 2.16. Acetyl-CoA is involved in the generation of the C6 mevalonate unit, a process that involves reduction by NADPH. Subsequent decarboxylation during phosphorylation (i.e. addition of phosphate) in the presence of ATP yields the fundamental isoprenoid unit, isopentenyl pyrophosphate (IPP), from which the terpenoids are synthesized by enzymatic condensation reactions. Recently, an alternative pathway has been discovered for the formation of IPP in various eubacteria and plants, which involves the condensation of glyceraldehyde 3-phosphate and pyruvate to form the intermediate 1-deoxy-D-xylulose 5-phosphate (Fig. 2.16 e.g. Eisenreich et al. 1998). We consider some of the more common examples of the main classes of terpenoids below. 

Enoch, H.G. and P. Strittmatter (1979). Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase. J. Biol. Chem. 254, 8976-8981. 

Guengerich, EP. and W.W. Johnson (1997). Kinetics of ferric cytochrome P450 reduction by NADPH-cytochrome P450 reductase Rapid reduction in the absence of substrate and variations among cytochrome P450 systems. Biochemistry 36, 14741-14750. 

Another consequence of the benzoquinone treatment concerns the oxygen sequence. In the untreated algae (Fig.2), the maximum yield is shifted to the 4-5 th flashes, compared to the 3rd flash in a regular sequence. This arises from the reduction of the plastoquinone pool that is known to occur in anaerobic algae. The state of this pool results from a balance between reduction by NADPH and oxidation by oxygen (chloro-... 

NADPH-Ferredoxin Reduces Heme b/j. The pathway of cyt be reduction by NADPH-ferredoxin is through the outside heme, since the reaction is not affected by the presence of the p-side inhibitor DBMIB. Therefore, the one-heme equivalent or 50% of the cyt be that is reduced must be either (i) the stromal-side heme, b, in all complexes, or (ii) a 1 1 mixture of fully oxidized and fully reduced complexes, the latter perhaps because of thylakoid membrane heterogeneity. These two possibilities can be tested since the amplitude of flash reduction would be smaller by a factor of two if half of the complexes are fully reduced, but unaffected if is initially reduced and bp oxidized in all complexes. The amplitude of cyt 6 reduction by a single turnover laser flash, measured in the absence and presence of NQNO, was not affected by pre-reduction with NADPH-Fd (Fig. 2A vs. 2B). This shows that there can be at most only a small fraction of 6-/complexes in which both hemes are reduced by NADPH-Fd, so that heme bn alone is reduced by NADPH-Fd in most complexes. 

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