The Role of Lysyl Residues in the Structure and Reactivity of Cytochrome b5 Reductase

Loverde, A. W.; Strittmatter, Philipp

doi:10.1016/s0021-9258(18)91932-0

Cited by 56 publications

(4 citation statements)

References 16 publications

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“…Site-Directed Mutagenesis of K110. On the basis of the acetylation studies of lysine residues and kinetic characterization of K110 mutants that either maintained or abolished positive charge, Strittmatter and co-workers suggested a role for K110 in NADH binding (8,19,25). However, in the crystal structure of pig cb5r, this residue was not located at the active site.…”

Section: Resultsmentioning

confidence: 99%

“…The microsomal form of cb5r has been isolated from a variety of sources, including human (6), rabbit (7), steer (8), rat (9), and porcine (10) liver, as either the mature amphipathic, detergent-solubilized form or the truncated, hydrophilic FAD-containing domain following limited proteolysis (11,12). In addition, various bacterial expression systems have been developed for the efficient production of the soluble functional forms of human (12), steer (13), and rat (14) liver cb5r.…”

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The Structure and Biochemistry of NADH-Dependent Cytochrome b₅ Reductase Are Now Consistent

2001

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Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 A resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 A resolution and in a complex with NAD+ at 2.3 A resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.

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Section: Resultsmentioning

confidence: 99%

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The Structure and Biochemistry of NADH-Dependent Cytochrome b₅ Reductase Are Now Consistent

2001

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“…In contrast to the structure of the wild-type enzyme, in the S127P variant, the majority of the interactions of the ADP moiety reside on the previously characterized NADH binding subdomain. The charge on the pyrophosphate group is neutralized by interactions with the Nζ atom of K110, which has previously been shown to be important in substrate binding (26,(32)(33)(34). The adenine moiety is housed in a 6 Å wide channel formed by residues P275-M277 in the NR4 helix on one side and F251 on the other.…”

Section: Resultsmentioning

confidence: 99%

The Structure of the S127P Mutant of Cytochrome b₅ Reductase that Causes Methemoglobinemia Shows the AMP Moiety of the Flavin Occupying the Substrate Binding Site

et al. 2003

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Methemoglobinemia, the first hereditary disease to be identified that involved an enzyme deficiency, has been ascribed to mutations in the enzyme cytochrome b(5) reductase. A variety of defects in either the erythrocytic or microsomal forms of the enzyme have been identified that give rise to the type I or type II variant of the disease, respectively. The positions of the methemoglobinemia-causing mutations are scattered throughout the protein sequence, but the majority of the nontruncated mutants that produce type II symptoms occur close to the flavin adenine dinucleotide (FAD) cofactor binding site. While X-ray structures have been determined for the soluble, flavin-containing diaphorase domains of the rat and pig enzymes, no X-ray or NMR structure has been described for the human enzyme or any of the methemoglobinemia variants. S127P, a mutant that causes type II methemoglobinemia, was the first to be positively identified and have its spectroscopic and kinetic properties characterized that revealed altered nicotinamide adenine dinucleotide hydride (NADH) substrate binding behavior. To understand these changes at a structural level, we have determined the structure of the S127P mutant of rat cytochrome b(5) reductase to 1.8 A resolution, providing the first structural snapshot of a cytochrome b(5) reductase mutant that causes methemoglobinemia. The high-resolution structure revealed that the adenosine diphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP binding site of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent with both the spectroscopic and kinetic data.

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“…While adding minimal bulk, the reaction preserves an amine’s physiological positive charge and, consequently, its electrostatic interactions. Studies have documented that amidination’s minimal invasiveness preserves the protein structure and enzymatic activity. − In terms of MS, the amidino group’s slight increase in p K a facilitates ionization …”

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In Vitro and In Vivo Chemical Labeling of Ribosomal Proteins: A Quantitative Comparison

et al. 2012

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Thioimidates have emerged as reagents for probing the protein structure, folding, and interactions under physiological conditions. The same properties that give thioimidates biological relevance make these molecules ideal candidates for use in vivo. Through labeling of ribosomal proteins, we have quantified the in vivo and in vitro reactivity of two thioimidates: S-methylthioacetimidate (SMTA) and a novel, charge-carrying analogue, S-sulfethylthioacetimidate (SSETA). In vitro experiments demonstrate that both amidinating reagents can probe the protein structure. Under comparable in vivo conditions, SMTA is found to be membrane-permeable while SSETA is not. The use of mass spectrometry with permeant and impermeant thioimidates promises insights into the membrane topology and protein structure in the native environment.

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The Role of Lysyl Residues in the Structure and Reactivity of Cytochrome b5 Reductase

Cited by 56 publications

References 16 publications

The Structure and Biochemistry of NADH-Dependent Cytochrome b₅ Reductase Are Now Consistent

The Structure and Biochemistry of NADH-Dependent Cytochrome b₅ Reductase Are Now Consistent

The Structure of the S127P Mutant of Cytochrome b₅ Reductase that Causes Methemoglobinemia Shows the AMP Moiety of the Flavin Occupying the Substrate Binding Site

In Vitro and In Vivo Chemical Labeling of Ribosomal Proteins: A Quantitative Comparison

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The Role of Lysyl Residues in the Structure and Reactivity of Cytochrome b5 Reductase

Cited by 56 publications

References 16 publications

The Structure and Biochemistry of NADH-Dependent Cytochrome b5 Reductase Are Now Consistent

The Structure and Biochemistry of NADH-Dependent Cytochrome b5 Reductase Are Now Consistent

The Structure of the S127P Mutant of Cytochrome b5 Reductase that Causes Methemoglobinemia Shows the AMP Moiety of the Flavin Occupying the Substrate Binding Site

In Vitro and In Vivo Chemical Labeling of Ribosomal Proteins: A Quantitative Comparison

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The Structure and Biochemistry of NADH-Dependent Cytochrome b₅ Reductase Are Now Consistent

The Structure and Biochemistry of NADH-Dependent Cytochrome b₅ Reductase Are Now Consistent

The Structure of the S127P Mutant of Cytochrome b₅ Reductase that Causes Methemoglobinemia Shows the AMP Moiety of the Flavin Occupying the Substrate Binding Site