Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

Durley, R. C. E.; Mathews, F.S.

doi:10.1107/s0907444995007827

Cited by 146 publications

(207 citation statements)

References 44 publications

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“…The structure solved in this work shows a 3/10 helix for residues 34-38, whereas residues 33 and 39 form hydrogen-bonded turns. As there is general agreement on the presence of an a-helix in this region [35], the above series of turns have been reported in Table 1 In the solution structure of oxidized bovine cytochrome h, these helices are 3/10 helices…”

Section: Structurementioning

confidence: 73%

“…The similarity (residues 3 -87) between microsomal rat cytochrome b, (which is the object of the present investigation) and the bovine isoenzyme is 94%, whereas the similarity between microsomal and mitochondrial rat cytochrome b, is 62%. A comparison of the present solution structure of reduced rat cytochrome b, with the crystal structure of oxidized bovine cytochrome b, [35] is shown in Fig. 5.…”

Section: Resultsmentioning

confidence: 99%

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Solution Structure of Reduced Microsomal Rat Cytochrome b₅

Banci

Bertini

Ferroni

et al. 1997

European Journal of Biochemistry

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The solution structure of the major form of the reduced soluble fragment of rat microsomal cytochrome b, has been solved through 'H-NMR spectroscopy. The protein contains 98 amino acids. Proton assignment was available for residues 1-94, except 90 [Guiles, R. D., Basus, V. J., Kuntz, I. D. & Waskell, L. (1992) Biochemistry 31, 11 365-11 3751 and has been confirmed. From 1722 NOES, of which 1203 were found to be meaningful, a family of 40 energy-minimized structures has been obtained with average backbone rmsd (for residues 5-89) of 0.078-tO.018 nm and average target function of 0.0045 nm', no distance violations being larger than 0.029 nm. The structure has been compared with the X-ray structure of the oxidized rat mitochondria1 isoenzyme and with that of the highly similar bovine microsomal isoenzyme in the oxidized form. The analysis of the elements of secondary structure is instructive in terms of their stability and of their occurrence in related structures, and of the capability of NMR and X-ray spectroscopy to observe them. Some detailed structural variations are noticed among the solved structures of the various isoenzymes and between solid and solution. The structural features in solution of the residues proposed to be involved in protein-protein recognition are found to be largely conserved with respect to the solid state.Keywords: cytochronie b,; protein recognition; solution structure; secondary structure.Cytochrome b, is a low-spin hemoprotein that acts as an electron-transfer mediator in numerous redox systems [I]. Different redox partners have been reported for cytochrome b, in vivo. In the process of fatty acid desaturation it interacts with NADH-cytochrome b, reductase [2, 31, and a role of cytochrome b, as electron donor to cytochrome P-450 has been proposed [4, 51. The protein is normally membrane bound, although a soluble form is found in erythrocytes, where its physiological role is that of reducing methemoglobin [6]. The membrane-bound form may be solubilized by incubation with proteolytic enzymes, such as trypsin. The soluble fragment of cytochrome b, still contains the heme and retains activity with respect to protein recognition and electron transfer [7]. It is well known that the soluble fragment of cytochrome b, interacts with cytochrome c, and that the reaction between the two is fast [7]. Such a complex has been an important model for the study of long-range biological electron transfer and, more generally, of the mechanisms of protein-protein recognition and interaction, mainly due because the crystallographic structures of the two individual components have been available since 1971 [8, 91. The interaction of cytochrome b, with cytochrome c has been studied extensively through a number of biochemical and biophysical methods [lo]. In particular, the small size of the two proteins allowed the investigation of the complex of the two through NMR spectroscopy as early as 1983 (111.The NMR investigation of cytochrome b, is significant in the frame of the current debate on the differences ...

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Section: Structurementioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Solution Structure of Reduced Microsomal Rat Cytochrome b₅

Banci

Bertini

Ferroni

et al. 1997

European Journal of Biochemistry

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“…4 in which the hydrophobic surface of cyt P450 is positioned in the outer bilayer of a membrane (34). Cyt b 5 has been manually docked with cyt P450 2C5 so that the cyt b 5 residues Asp 65 and Val 66 are near residues Lys 121 (at the beginning of the C helix) and Lys 429 of cyt P450 2C5 (8,31,34 If the 15 amino acids of the linker were extended like a parallel ␤-sheet, its maximum length would be Х51 Å (3.5 Å/residue), whereas if arranged as an ␣-helix, the 15 residues would span merely Х22.5 Å (1.5 Å/amino acid) (37). A 7-amino acid linker would therefore be predicted to span 24 Å if maximally extended but only 10.5 Å if folded as an ␣-helix.…”

Section: Construction and Expression Of Mutant Cyts B 5 With Linker Rmentioning

confidence: 99%

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Clarke

Bidwai

et al. 2004

Journal of Biological Chemistry

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Cytochrome b 5 (cyt b 5 ) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane ␣-helix at its carboxyl terminus. These two domains are connected by an ϳ15-amino acid linker domain, Ser 90 -Asp 104 , which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b 5 to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b 5 to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b 5 except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b 5 to interact with cyt P450 2B4. A model for the membranebound cyt b 5 -cyt P450 complex is presented.Microsomal cytochrome b 5 (cyt b 5 ) 1 is an amphipathic electron transfer hemoprotein located in the membrane of the endoplasmic reticulum. It provides electrons for a broad range of reactions, including fatty acid desaturation, cholesterol biosynthesis, and a variety of cytochrome P450-dependent oxidation and hydroxylation reactions (1, 2).Cytochrome P450 (cyt P450) is responsible for the oxidation of a large number of substrates. The overall stoichiometry of the reaction catalyzed by cyt P450 is shown in Reaction 1, where RH is the substrate (3).The cyt P450 catalytic reaction occurs via a reaction cycle that involves substrate binding, reduction of the ferric heme, O 2 binding, reduction of the oxyferrous heme (second electron transfer), substrate oxidation, and finally product dissociation (4). Both ferric and oxyferrous cyt P450 receive electrons from NADPH via its redox partner, NADPH-cytochrome P450 reductase (cyt P450 reductase), whereas cyt b 5 can donate the second electron. Because of the high redox potential of Х20 mV for cyt b 5 , the first electron required to reduce cyt P450 from the ferric to the ferrous state is always transferred from NADPH via cyt P450 reductase. However, as the redox potential of cyt P450 increases from ХϪ230 mV to ϩ50 mV on transition to the oxyferrous state, the second electron can be obtained from either cyt P450 reductase or cyt b 5 (5, 6).Depending on the substrate, isozyme of cyt P450 and the experimental conditions cyt b 5 can stimulate, inhibit, or have no effect on cyt P450 activity (2). Studies of the stoichiometry of the metabolism of benzphetamine and methoxyflurane by cyt P450 2B4 have shown that cyt b 5 increases the efficiency of catalysis by cyt P450 2B4 primarily by decreasing the formation of the side-product superoxide. However, cyt b...

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“…Other heme proteins with bis-histidyl coordination do not bind ligands [109,110], and a hexacoordinate Mb mutant protein is structurally constrained, and limited in its ability to bind exogenous ligands [53]. Thus, there has been interest in observing the structural changes accompanying ligand binding in the context of naturally occurring hxHbs.…”

Section: Structures Of Hexacoordinate Hemoglobinsmentioning

confidence: 99%

Structure and Reactivity of Hexacoordinate Hemoglobins

Hargrove

Sturms

2013

Free Radical Biology and Medicine

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The term "hemoglobin" has spent decades at the forefront of biochemical research, including the eras of "grind-and-find", protein structure determination, and the relationship between protein structure and function. It has also served as a familiar target in genomic annotation, and as a guidepost to study the evolution of primary structure and protein function. We have learned over the past fifteen years that most organisms contain genes with homology to globins, and that not all glo- AbstractThe heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called "pentacoordinate" hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called "hexacoordinate hemoglobins", which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal.

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Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

Cited by 146 publications

References 44 publications

Solution Structure of Reduced Microsomal Rat Cytochrome b₅

Solution Structure of Reduced Microsomal Rat Cytochrome b₅

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Structure and Reactivity of Hexacoordinate Hemoglobins

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Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

Cited by 146 publications

References 44 publications

Solution Structure of Reduced Microsomal Rat Cytochrome b5

Solution Structure of Reduced Microsomal Rat Cytochrome b5

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Structure and Reactivity of Hexacoordinate Hemoglobins

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Solution Structure of Reduced Microsomal Rat Cytochrome b₅

Solution Structure of Reduced Microsomal Rat Cytochrome b₅