Computer-assisted docking of flavodoxin with the ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein-protein interactions but not for catalysis

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA.FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA.FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.     

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar typhimurium LT2

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH(2) dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH(2) occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH(2) in the reaction mixture. FMNH(2) failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.     

Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium.

The cobA gene of Salmonella typhimurium and its product were overexpressed to approximately 20% of the total cell protein. CobA was purified to 98% homogeneity; N-terminal sequence analysis (21 residues) of homogeneous protein confirmed the predicted amino acid sequence. ATP:corrinoid adenosyltransferase activity was demonstrated in vitro to be associated with CobA. This activity was optimal at pH 8 and 37 degrees C. A quantitative preference was determined for Mn(II) cations and ATP. The apparent Km of CobA for ATP was 2.8 microM, and that for cob(I)alamin was 5.2 microM. Vmax was measured at 0.43 nmol/min. Cobinamide served as the substrate for CobA to yield adenosylcobinamide. Activity was stable at 4 degrees C for several weeks but was lost rapidly at room temperature (50% overnight). Dithiothreitol was required to maintain the enzymatic activity of CobA.     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain G?1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.     

The ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2'-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct

The specificity of the ATP:corrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica serovar Typhimurium LT2 for its nucleotide substrate was tested using ATP analogs and alternative nucleotide donors. The enzyme showed broad specificity for the nucleotide base and required the 2'-OH group of the ribosyl moiety of ATP for activity. (31)P NMR spectroscopy was used to identify inorganic triphosphate (PPP(i)) as the byproduct of the reaction catalyzed by the CobA enzyme. Cleavage of triphosphate into pyrophosphate and orthophosphate did not occur, indicating that triphosphate cleavage was not required for release of the adenosylcorrinoid product. Triphosphate was a strong inhibitor of the reaction, with 85% of CobA activity lost when the ATP/PPP(i) ratio present in the reaction mixture was 1:2.5.     

Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP

In Salmonella typhimurium, formation of the cobalt-carbon bond in the biosynthetic pathway for adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196 amino acid residues. This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosyl moiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide, its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin. Three X-ray structures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and a ternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 A resolution, respectively. These structures show that the enzyme is a homodimer. In the apo structure, the polypeptide chain extends from Arg(28) to Lys(181) and consists of an alpha/beta structure built from a six-stranded parallel beta-sheet with strand order 324516. The topology of this fold is very similar to that seen in RecA protein, helicase domain, F(1)ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase where a P-loop is located at the end of the first strand. Strikingly, the nucleotide in the MgATP.CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases. That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate. The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate. In the ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depression at the C-terminal end of the beta-sheet of one subunit; however, the active site is capped by the N-terminal helix from the symmetry-related subunit that now extends from Gln(7) to Ala(24). The lower ligand of cobalamin is well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit. Interestingly, there are few interactions between the protein and the polar side chains of the corrin ring which accounts for the broad specificity of this enzyme. The corrin ring is oriented such that the cobalt atom is located approximately 6.1 A from C5' of the ribose and is beyond the range of nucleophilic attack. This suggests that a conformational change occurs in the ternary complex when Co(III) is reduced to Co(I).     

Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, kcat = 0.06 s(-1), and Vmax = 105 nm min(-1). Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was > or =50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the beta-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.     

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans.

Cob(I)alamin adenosyltransferase (EC 2.5.1.17) was purified to homogeneity from extracts of a Pseudomonas denitrificans recombinant strain and sequenced at its N terminus. It is a homodimer (each unit with an Mr of 28,000) encoded by cobO. The enzyme adenosylated all of the corrinoids isolated from this microorganism but did not adenosylate cobyrinic acid.     

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi⁺). This process requires the formation of “supernucleophilic” Co(I)rrinoid intermediates in the enzyme active site which are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.     

Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase

The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.     

Evidence that a B12-adenosyl transferase is encoded within the ethanolamine operon of Salmonella enterica

Adenosylcobalamin (Ado-B12) is both the cofactor and inducer of ethanolamine ammonia lyase (EA-lyase), a catabolic enzyme for ethanolamine. De novo synthesis of Ado-B12 by Salmonella enterica occurs only under anaerobic conditions. Therefore, aerobic growth on ethanolamine requires import of Ado-B12 or a precursor (CN-B12 or OH-B12) that can be adenosylated internally. Several known enzymes adenosylate corrinoids. The CobA enzyme transfers adenosine from ATP to a biosynthetic intermediate in de novo B12 synthesis and to imported CN-B12, OH-B12, or Cbi (a B12 precursor). The PduO adenosyl transferase is encoded in an operon (pdu) for cobalamin-dependent propanediol degradation and is induced by propanediol. Evidence is presented here that a third transferase (EutT) is encoded within the operon for ethanolamine utilization (eut). Surprisingly, these three transferases share no apparent sequence similarity. CobA produces sufficient Ado-B12 to initiate eut operon induction and to serve as a cofactor for EA-lyase when B12 levels are high. Once the eut operon is induced, the EutT transferase supplies more Ado-B12 during the period of high demand. Another protein encoded in the operon (EutA) protects EA-lyase from inhibition by CN-B12 but does so without adenosylation of this corrinoid.     

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.     

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The defect in the cblB class of human methylmalonic acidemia: Deficiency of cob(I)alamin adenosyltransferase activity in extracts of cultured fibroblasts

We show that the reductants present in the $\text{in}\text{vitro}$ assay used to measure the formation of adenosylcobalamin from cob(III)alamin by cell-free extracts of human fibroblasts result in the non-enzymatic reduction of cob(III)alamin to cob(I)alamin. Hence, the $\text{in}\text{vitro}$ assay uniquely estimates the activity of ATP:cob(I)alamin adenosyltransferase (EC 2.5.1.17). Based on additional studies with extracts of fibroblasts from patients in the $\text{cbl}\text{B}$ class of human methylmalonic acidemia and from their parents, we conclude that this mutant class results from a specific deficiency of adenosyltransferase activity which is inherited as an autosomal recessive trait.     

Functional characterization and mutation analysis of human ATP:Cob(I)alamin adenosyltransferase

ATP:cob(I)alamin adenosyltransferase catalyzes the final step in the conversion of vitamin B 12 into the active coenzyme, adenosylcobalamin. Inherited defects in the gene for the human adenosyltransferase (hATR) result in methylmalonyl aciduria (MMA), a rare but life-threatening illness. In this study, we conducted a random mutagenesis of the hATR coding sequence. An ATR-deficient strain of Salmonella was used as a surrogate host to screen for mutations that impaired hATR activity in vivo. Fifty-seven missense mutations were isolated. These mapped to 30 positions of the hATR, 25 of which had not previously been shown to impair enzyme activity. Kinetic analysis and in vivo tests for enzyme activity were performed on the hATR variants, and mutations were mapped onto a hATR structural model. These studies functionally defined the hATR active site and tentatively implicated three amino acid residues in facilitating the reduction of cob(II)alamin to cob(I)alamin which is a prerequisite to adenosylation.     

Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein

ATP:Cobalamin adenosyltransferases catalyze the transfer a 5′-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co–C bond of coenzyme B₁₂. There are three types of adenosyltransferases, CobA, PduO and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases is the most widely distributed enzyme. Structural comparisons between apo BcPduO and BcPduO in complex with MgATP revealed that the N-terminal strands of both structures were ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Furthermore, unlike other reported structures, apo BcPduO was bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site. This study provides more structural information into the role of active site residues on substrate binding.     

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.     

Ligand trans influence governs conformation in cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a large, modular enzyme that uses a cobalamin prosthetic group as a donor or acceptor in three separate methyl transfer reactions. The prosthetic group alternates between methylcobalamin and cob(I)alamin during catalysis as homocysteine is converted to methionine using a methyl group derived from methyltetrahydrofolate. Occasional oxidation of cob(I)alamin to cob(II)alamin inactivates the enzyme. Reductive methylation with flavodoxin and adenosylmethionine returns the enzyme to an active methylcobalamin state. At different points during the reaction cycle, the coordination state of the cobalt of the cobalamin changes. The imidazole side chain of His759 coordinates to cobalamin in a "His-on" state and dissociates to produce a "His-off" state. The His-off state has been associated with a conformation of MetH that is poised for reactivation of cobalamin by reductive methylation rather than catalysis. Our studies on cob(III)alamins bound to MetH, specifically aqua-, methyl-, and n-propylcobalamin, show a correlation between the accessibility of the reactivation conformation and the order of the established ligand trans influence. The trans influence also controls the affinity of MetH in the cob(III)alamin form for flavodoxin. Flavodoxin, which acts to shift the conformational equilibrium toward the reactivation conformation, binds less tightly to MetH when the cob(III)alamin has a strong trans ligand and therefore has less positive charge on cobalt. These results are compared to those for cob(II)alamin MetH, illustrating that access to the reactivation conformation is governed by the net charge on the cobalt as well as the trans influence in cob(III)alamins.     

The ferredoxin-NADP(H) reductase from Rhodobacter capsulatus: molecular structure and catalytic mechanism

The photosynthetic bacterium Rhodobacter capsulatus contains a ferredoxin (flavodoxin)-NADP(H) oxidoreductase (FPR) that catalyzes electron transfer between NADP(H) and ferredoxin or flavodoxin. The structure of the enzyme, determined by X-ray crystallography, contains two domains harboring the FAD and NADP(H) binding sites, as is typical of the FPR structural family. The FAD molecule is in a hairpin conformation in which stacking interactions can be established between the dimethylisoalloxazine and adenine moieties. The midpoint redox potentials of the various transitions undergone by R. capsulatus FPR were similar to those reported for their counterparts involved in oxygenic photosynthesis, but its catalytic activity is orders of magnitude lower (1-2 s(-)(1) versus 200-500 s(-)(1)) as is true for most of its prokaryotic homologues. To identify the mechanistic basis for the slow turnover in the bacterial enzymes, we dissected the R. capsulatus FPR reaction into hydride transfer and electron transfer steps, and determined their rates using stopped-flow methods. Hydride exchange between the enzyme and NADP(H) occurred at 30-150 s(-)(1), indicating that this half-reaction does not limit FPR activity. In contrast, electron transfer to flavodoxin proceeds at 2.7 s(-)(1), in the range of steady-state catalysis. Flavodoxin semiquinone was a better electron acceptor for FPR than oxidized flavodoxin under both single turnover and steady-state conditions. The results indicate that one-electron reduction of oxidized flavodoxin limits the enzyme activity in vitro, and support the notion that flavodoxin oscillates between the semiquinone and fully reduced states when FPR operates in vivo.