Molecular Dissection of Human Methionine Synthase Reductase:  Determination of the Flavin Redox Potentials in Full-Length Enzyme and Isolated Flavin-Binding Domains

Wolthers, Kirsten R.; Basran, Jaswir; Munro, Andrew W.; Scrutton, Nigel S.

doi:10.1021/bi027290b

Cited by 52 publications

(77 citation statements)

References 36 publications

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“…These values are in agreement with the redox potentials obtained from this work for the FMN of the soluble diflavin WT rat CYPOR (Ϫ56 mV for FMNox/sq and Ϫ249 mV for FMN sq/hq) at pH 7.4. Previous experiments on diflavin reductases have shown that the potentials of the FMN domain are similar in the diflavin and separate flavin domains (29,58,59). Because of the confounding presence of both FAD and FMN in the diflavin CYPOR, the FMN redox potentials could not be unambiguously assigned for the diflavin ⌬Gly-143 and two ⌬Gly-141 mutants.…”

Section: Determination Of the Redox Potentials Of The Fmn Domainsmentioning

confidence: 94%

Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Rwere

Xia

et al. 2016

Journal of Biological Chemistry

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NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochromes P450 via its FAD and FMN.To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type and mutant structures reveals that the basis for the air stability of the neutral blue semiquinone is protonation of the flavin N5 and strong H-bond formation with the Gly-141 carbonyl. The ⌬Gly-143 protein had moderately decreased activity with cytochrome P450 and cytochrome c. It formed a flexible loop, which transiently interacts with the flavin N5, resulting in the generation of both an unstable neutral blue semiquinone and hydroquinone. The ⌬Gly-141 and ⌬G141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c. In the ⌬Gly-141 mutants, the backbone amide of Glu/Asn-142 forms an H-bond to the N5 of the oxidized flavin, which leads to formation of an unstable red anionic semiquinone with a more negative potential than the hydroquinone. The semiquinone of ⌬G141/E142N was slightly more stable than that of ⌬Gly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both ⌬Gly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (⌬Gly-810). Our results indicate that the catalytic activity of cytochrome P450 oxidoreductase is a function of the length, sequence, and flexibility of the 140s loop and illustrate the sophisticated variety of biochemical mechanisms employed in fine-tuning its redox properties and function.

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Section: Determination Of the Redox Potentials Of The Fmn Domainsmentioning

confidence: 94%

Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Rwere

Xia

et al. 2016

Journal of Biological Chemistry

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“…Extensive studies of the interactions of purified hMSR with NADPH have been performed (15)(16)(17). However, overexpression of an active recombinant hMS has heretofore not been achieved, preventing biochemical studies of the interactions between hMS and hMSR.…”

mentioning

confidence: 99%

Human methionine synthase reductase is a molecular chaperone for human methionine synthase

Yamada

Gravel

Toraya

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a K act value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the presence of E. coli flavodoxin and flavodoxin reductase, which maintain the activity of E. coli MS. These results indicate that recognition of MS by their reductive partners is very strict, despite the high homology between MS from different species. The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS). In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced. The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37°C. MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme. Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin reductase, rather than acting solely in the reductive activation of MS.aquacobalamin reductase ͉ cobalamin ͉ holoenzyme formation

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“…The eukaryotic NOS isoforms, like P450 BM3, contain heme b bound to an oxygenase domain covalently linked to the reductase domain (18). In recent work, the reduction potentials for each of the couples of the flavin cofactors in P450 BM3, neuronal NOS, human CPR, human novel reductase 1, and methionine synthase reductase have been determined by anaerobic spectroelectrochemistry (6,10,(17)(18)(19).…”

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confidence: 99%

Switching Pyridine Nucleotide Specificity in P450 BM3

Neeli

Roitel²,

Scrutton

et al. 2005

Journal of Biological Chemistry

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Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k cat /K m (NADPH)/k cat /K m (NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wildtype reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a chargetransfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH 2 -NAD(P) ؉ charge-transfer species, suggesting a relatively slow rate of release of NAD(P)؉ from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/ semiquinone couple, and a concomitant ϳ100-mV elevation in the 2-electron redox couple for the enzymebound FAD (؊320, ؊220, and ؊224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).Bacillus megaterium flavocytochrome P450 BM3 is a high activity fatty acid hydroxylase enzyme that has evolved from fusion of a eukaryotic-like NADPH-cytochrome P450 reductase (CPR) 1 to a P450 in a single polypeptide (1). The fusion provides an efficient electron transport system from NADPH, through the FAD and FMN cofactors in the CPR, and onto the heme b in the P450 (2). P450 BM3 has the highest monooxygenase activity of any P450 system examined to date (3). Molecular dissection has been used to generate the individual heme and CPR domains for structural, thermodynamic, and kinetic studies (e.g. Refs. 4 -7). The component flavodoxin-like (FMN-containing) and ferredoxin reductase-like (FAD-containing) domains of the CPR have also been generated, supporting the hypothesis that the enzyme has a discrete domain organization and that the BM3 CPR and other related enzymes evolved by fusion of genes encoding the smaller flavoproteins (8). Molecular dissection of the related human CPR to produce component flavin-binding domains has been reported (9). The isolated domains of human CPR exchange electrons and have thermodynamic properties almost identical to those of intact CPR (9, 10). The availability of isolated domains has enabled detailed study of electron transfer mechanisms in wild-type and mutant proteins (11,12).Fla...

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Molecular Dissection of Human Methionine Synthase Reductase: Determination of the Flavin Redox Potentials in Full-Length Enzyme and Isolated Flavin-Binding Domains

Cited by 52 publications

References 36 publications

Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Human methionine synthase reductase is a molecular chaperone for human methionine synthase

Switching Pyridine Nucleotide Specificity in P450 BM3

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