scholarly journals Are plastocyanin and ferredoxin specific electron carriers or generic redox capacitors? Classical and murburn perspectives on two chloroplast proteins

2020 ◽  
Author(s):  
Daniel Andrew Gideon ◽  
Vijay Nirusimhan ◽  
Kelath Murali Manoj
Keyword(s):  
Classical Model ◽  
Cytochrome B5 ◽  
Affinity Binding ◽  
Amino Acid Residues ◽  
Light Reaction ◽  
Mutant Proteins ◽  
Chloroplast Proteins ◽  
Redox Partners ◽  
Kd Values ◽  
Specific Affinity

Within the context of light reaction of photosynthesis, the structure-function correlations of the chloroplast proteins of plastocyanin and ferredoxins (Fd) are analyzed via two perspectives: 1) The Z-scheme, which considers PC/Fd as specific affinity binding-based electron-relay agents, thereby deterministically linking the functions of Cytochrome b6f (Cyt. b6f) and Photosystem I (PS I) to NADP+ reduction by Fd:NADPH oxidoreductase (FNR) via protein-protein contacts and 2) The murburn explanation for oxygenic photophosphorylation, which deems PC/Fd as generic ‘redox capacitors’, temporally accepting and releasing one-electron equivalents in reaction milieu. Amino acid residues located on the surface loci of key patches of PC/Fd vary in electrostatic/contour (topography) signatures. Crystal structures of four different complexes each of cyt.f-PC and Fd-FNR show little conservation in the contact-surfaces, thereby discrediting ‘affinity binding-based electron transfers (ET)’ as an evolutionary logic. Further, thermodynamic and kinetic data on wildtype and mutant proteins interactions do not align well with model 1. Furthermore, micromolar physiological concentrations of PC (when Kd values 100 μM) and the non-conducive architecture of chloroplasts render the classical model untenable. In the 2nd model, PC is optional and higher concentrations of PC (sought by model 1) could inhibit ET, quite like the role of cytochrome c of mitochondria and cytochrome b5 of cytoplasmic microsomes. Also, PC is found in both lumen and stroma, and plants lacking PC survive and grow. Thus, evidence from structure, interactive dynamics with redox partners and physiological implications of PC/Fd supports the murburn perspective that these proteins serve as generic redox-capacitors in chloroplasts.

scholarly journals Roles of cytochromes c and b5 in mitochondria and microsomes: Classical and murburn perspectives

2020 ◽  
Author(s):  
Kelath Murali Manoj ◽  
Daniel Andrew Gideon
Keyword(s):  
Protein Interactions ◽  
Cytochrome B5 ◽  
Specific Interaction ◽  
Complex Iii ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Redox Equilibrium ◽  
Redox Partners ◽  
Cyt C ◽  
Altered Surface

While cytochrome c (Cyt. c) is a soluble protein involved in mitochondrial electron transfer (ET) reactions between Complex III and Complex IV, cytochrome b5 (Cyt. b5) is a microsomal membrane protein acting as a redox aide for diverse cytochrome P450s and their unique reductase. We found little conservation in the sequence and surface amino acid residues of Cyt. c and b5 proteins among evolutionarily diverse species. Hence, the logic that these proteins mediate ET through affinity binding via specific surface residues is weak. Also, analysis of putative protein-protein interactions in the crystal structures of these proteins and their redox partners did not point to any specific interaction logic. The literature on kinetic and thermodynamic constants of mutants (with altered surface residues) did not provide strong evidence to support the binding-based ET paradigm. Topographically divergent Cyt. b5 from one species has been shown to enhance the activity of CYP450 from another species, implying the involvement of non-specific interactions. These observations downplay the classical protein-protein biding based long range ET mechanism. Further, we provide evidence to show that murburn concept presents better chemico-physical logic for ETs mediated by Cyt. c and b5. To explain for the promiscuity of interactions, we conclude that the two proteins act as non-specific/generic redox capacitors, mediating a one-electron redox equilibrium involving diffusible reactive oxygen species (DROS) and ions.

scholarly journals Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.

1995 ◽  
Vol 15 (10) ◽  
pp. 5635-5644 ◽  
Author(s):  
Y Liu ◽  
N Mathias ◽  
C N Steussy ◽  
M G Goebl
Keyword(s):  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Cloning And Sequencing ◽  
Mutant Proteins ◽  
Catalytic Domains ◽  
Conserved Regions ◽  
E2 Enzymes ◽  
Key Residues

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

scholarly journals Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

2009 ◽  
Vol 90 (7) ◽  
pp. 1730-1733 ◽  
Author(s):  
Natalia L. Varich ◽  
Konstantin S. Kochergin-Nikitsky ◽  
Evgeny V. Usachev ◽  
Olga V. Usacheva ◽  
Alexei G. Prilipov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Antigenic Variation ◽  
Strain Differences ◽  
Antigenic Structure ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Antigenic Sites

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

scholarly journals Four Amino Acid Residues Are Critical for High Affinity Binding of Neuromedin B to the Neuromedin B Receptor

1998 ◽  
Vol 273 (26) ◽  
pp. 15927-15932 ◽  
Author(s):  
Eduardo Sainz ◽  
Mark Akeson ◽  
Samuel A. Mantey ◽  
Robert T. Jensen ◽  
James F. Battey
Keyword(s):  
Amino Acid ◽  
Affinity Binding ◽  
Amino Acid Residues ◽  
High Affinity Binding ◽  
High Affinity ◽  
Neuromedin B

The direct electrochemistry of cytochrome b5 and its mutant proteins

1997 ◽  
Vol 428 (1-2) ◽  
pp. 39-45 ◽  
Author(s):  
Yun-Hua Wang ◽  
Jun Cui ◽  
Yu-Long Sun ◽  
Ping Yao ◽  
Ji-Hua Zhuang ◽  
...  
Keyword(s):  
Cytochrome B5 ◽  
Direct Electrochemistry ◽  
Mutant Proteins

scholarly journals Substrate (aglycone) specificity of human cytosolic beta-glucosidase

2003 ◽  
Vol 373 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Jean-Guy BERRIN ◽  
Mirjam CZJZEK ◽  
Paul A. KROON ◽  
W. Russell MCLAUCHLAN ◽  
Antoine PUIGSERVER ◽  
...  
Keyword(s):  
Structural Model ◽  
Homology Modelling ◽  
Three Dimensional ◽  
Amino Acid Residues ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Mutant Proteins ◽  
Metabolizing Enzyme ◽  
Beta Glucosidase

Human cytosolic β-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays β-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (β/α)8-barrel fold characteristic of family 1 β-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val168→Tyr, Phe225→Ser, Tyr308→Ala and Tyr308→Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val168→Tyr mutation did not affect Km on p-nitrophenyl (pNP)-glycosides, but increased Km 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe225→Ser and Tyr308→Ala mutations, and, to a lesser degree, the Tyr308→Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.

scholarly journals Mapping of catalytically important residues in the rat l-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches

2004 ◽  
Vol 379 (2) ◽  
pp. 253-261 ◽  
Author(s):  
John V. FLEMING ◽  
Francisca SÁNCHEZ-JIMÉNEZ ◽  
Aurelio A. MOYA-GARCÍA ◽  
Michael R. LANGLOIS ◽  
Timothy C. WANG
Keyword(s):  
Vitamin B6 ◽  
Histidine Decarboxylase ◽  
Site Directed Mutagenesis ◽  
Dopa Decarboxylase ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Evolutionarily Conserved ◽  
Mutagenesis Study ◽  
Structural Levels ◽  
Homologous Enzyme

HDC (l-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from l-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme l-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an l-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.

Sign in / Sign up

Export Citation Format

Share Document