Nitrogen Fixation. BiologicalSymbiotic Non Symbiotic AbiologicalIndustrialNatural

Nitrogen Fixation

BiologicalSymbiotic Non Symbiotic AbiologicalIndustrialNatural

Abiological nitrogen fixation In abiological nitrogen fixation the nitrogen is reduced to ammonia without involving any living cell. Abiological fixation can be of two types : industrial and natural. For example, in the Habers process, synthetic ammonia is produced by passing a mixture of nitrogen and hydrogen through a bed of catalyst (iron oxides) at a very high temperature and pressure. In natural process nitrogen can be fixed especially during electrical discharges in the atmosphere. It may occur during lightning storms and nitrogen in the atmosphere can combine with oxygen to form oxides of nitrogen.

Biological Nitrogen Fixation Symbiotic Root nodule Formation Enzyme ActionNon Symbiotic Biological nitrogen fixation Conversion of Atmospheric nitrogen (N=N) is reduced to ammonia in the presence of nitrogenase.

Nitrogenase is a biological catalyst found in some microorganisms. such as the symbiotic Rhizobium and Frankia, or the free-living Azospirillum and Azotobacter. Biological nitrogen fixation is brought about both by free-living soil microorganisms and by symbiotic associations of microorganisms with higher plants. Leguminous plants fix atmospheric nitrogen by working symbiotically with special bacteria, rhizobia, which live in the root nodules. Rhizobia infect root hairs of the leguminous plants and produce the nodules. The nodules become the home for bacteria where they obtain energy from the host plant and take free nitrogen from the soil air and process it into combined nitrogen. In return, the plant receives the fixed N from nodules and produces food and forage protein.

Mechanism of biological nitrogen fixation Nitrogen fixation requires: i.The molecular nitrogen ii.A strong reducing power to reduce nitrogen like FAD (Flavin adenine Dinucleotide iii.Source of energy (ATP) to transfer hydrogen atoms to dinitrogen and iv.Enzymes nitrogenase and Legheamoglobin v.Compound for trapping the ammonia formed since it is toxic to cells. vi.The reducing agent and ATP are provided by photosynthesis and respiration.

Symbiotic Biological Nitrogen Fixation : Root Nodule Formation

Hemoglobin OXYGEN TRANSPORT

Proteins and their Relation to life DNARNAPROTEIN STRUCTUREANTIBODIES STORAGE TRANSPORT ENZYMES

Myoglobin, Hemoglobin, Cytochromes bind O2 Oxygen is transported from lungs to various tissues via blood in association with hemoglobin In muscle, hemoglobin gives up O2 to myoglobin which has a higher affinity for O2 than hemoglobin. Oxygen-binding curve for hemoglobin is sigmoidal whereas for myoglobin it is hyperbolic. This facilitates transfer of O2 to myoglobin. Cytochromes participate is redox reactions and are components of the electron transport chain. Oxygen-Binding Proteins

Hemoglobin is a O 2 transport protein found in the RBCs An oligomeric protein made up of 2 dimers, a total of 4 polypeptide chains: 1122. Molecular weight : 64,500. The (141 aa) and (146 aa) subunits have < 50 % identity. The 3D- structures of (141 aa) and (146 aa) subunits of hemoglobin and the single polypeptide of myoglobin are very similar; all three are members of the globin family. Each Hb subunit consists of 7 () or 8 () alpha helices and several bends and loops folded into a single globin domain. Each subunit has a heme-binding pocket. Hemoglobin Structure

The heme group is responsible for the O 2 -binding capacity of hemoglobin. The heme group consists of the planar aromatic protoporphyrin made up of four pyrrole rings linked by methane bridges. A Fe atom in its ferrous state (Fe +2 ) is at the center of protoporphyrin. Fe +2 has 6 coordination bonds, four bonded to the 4 pyrrole N atoms. The nucleophilic N prevent oxidation of Fe +2. The two additional binding sites are one on either side of the heme plane. One of these is occupied by the imidazole group of His. The second site can be reversibly occupied by O 2, which is hydrogen bonded to another His. The Prosthetic Heme Group

When hemoglobin is bound to O 2, it is called oxyhemoglobin. This is the relaxed (R ) state. The form with a vacant O 2 binding site is called deoxyhemoglobin and corresponds to the tense (T) state. If iron is in the oxidized state as Fe +3, it is unable to bind O 2 and this form is called as methemoglobin CO and NO have higher affinity for heme Fe +2 than O 2 and can displace O 2 from Hb, accounting for their toxicity. Different forms of Hemoglobin

Hemoglobin exists in two major conformational states: Relaxed (R ) and Tense (T) R state has a higher affinity for O2. In the absence of O2, T state is more stable When O2 binds, R state is more stable, so hemoglobin undergoes a conformational change to the R state. The structural change involves readjustment of interactions between subunits. T and R states of Hemoglobin

O2 binding rearranges electrons within Fe+2 making it more compact so that it fits snugly within the plane of porphyrin. Since Fe is bound to histidine of the globin domain, when Fe moves, the entire subunit undergoes a conformational change. This causes hemoglobin to transition from the tense (T) state to the relaxed (R) state. The 11 and 22 dimers rearrange and rotate approximately 15 degrees with respect to each other Inter-subunit interactions influence O2 binding to all 4 subunits resulting in cooperativity. Changes Induced by O2 Binding

Four subunits, so four O2-binding sites O2 binding is cooperative meaning that each subsequent O2 binds with a higher affinity than the previous one Similarly, when one O2 is dissociated, the other three will dissociate at a sequentially faster rate. Due to positive cooperativity, a single molecule is very rarely partially oxygenated. There is always a combination of oxygenated and deoxygenated hemoglobin molecules. The percentage of hemoglobin molecules that remain oxygenated is represented by its oxygen saturation. O2-binding curves show hemoglobin saturation as a function of the partial pressure for O2. O2-binding kinetics

Myoglobin OXYGEN TRANSPORT

A simple oxygen-binding protein found in almost all mammals, primarily in muscle tissue. Myoglobin (Mr 16,700; abbreviated Mb) As a transport protein, it facilitates oxygen diffusion in muscle. Myoglobin is abundant in the muscles of diving mammals such as seals and whales Also has oxygen storage function for prolonged excursions undersea. A single polypeptide of 153 amino acid residues with one molecule of heme. It is typical of the family of proteins called globins, all of which have similar primary and tertiary structures. The polypeptide is made up of eight -helical segments(78%) connected by bends. Myoglobin - Structure

Chlorophyll PORPHYRINES

Porphyrins (pronounce) are tetrapyrroles. They consist of four weakly aromatic pyrrole (pronounce) rings joined by methene bridges. Porphyrin is a heterocyclic macrocycle made from 4 pyrrole subunits linked on opposite sides ( position) through 4 methinebridges (=CH-). The extensive conjugated system makes the compound chromatic, hence the name porphyrin, from a Greek word for purple The macrocycle has 22 pi electrons, 18 of which are active in the conjugated system. These are the central groups of biologically imp molecules such as Hemoglobin, Myoglobin, Chlorophyll, Cytochromes, etc Porphyrins

Because of the unique chemistry of porphyrins, they are able to perform Several functions: As a metal binder (ligands) As a solar cell (convert light or chemical energy) As an oxygen transport medium (hemoglobin) As an electron transfer medium (conducting polymers) Gene regulation Drug metabolism Iron metabolism Hormone synthesis Uses of Porphyrins

As the basic building block of hemoglobin Heme a cross-coupled porphyrin used in the larger molecule hemoglobin

Cytochrome C a molecule responsible for transporting an electron used to provide energy to the organism. These molecules are identical, or very similar, for related species of plants or animals.

A green compound found in leaves and green stems of plants. It occurs in cell organelles called chloroplasts, which are absent in animals. Chlorophyll is the molecule that traps this 'most elusive of all powers and is called a photoreceptor. The basic structure of a chlorophyll molecule is a porphyrin ring, coordinated to a central atom. This is very similar in structure to the heme group found in hemoglobin, except that in heme the central atom is iron, whereas in chlorophyll it is magnesium. Chlorophyll

Chlorophyll a and chlrophyll b The most important pigment in plants is chlorophyll. Two types of chlorophyll in plants, chlorophyll a (chl a) and chlorophyll b (chl b) Chlorophyll is composed of two parts; the first is a porphyrin ring with magnesium at its center, the second is a hydrophobic phytol tail The ring has many delocalized electrons that are shared between several of the C, N, and H atoms; these delocalized electrons are very important for the function of chlorophyll. The tail is a 20 carbon chain that is highly hydrophic and stabilizes the molecule in the hydrophobic core of the thylakoid membrane.

Structurally CH 3 group is present in chl a where chl b has a CHO group. Chlorophyll a and b absorb different wavelengths better than others. chl a absorbs best at 450 and 680 nm chl b absorbs best at 500 and 640 nm chlorophyll a is directly involved in the redox reactions of the light reactions, chl b functions as an accessory pigment Accessory pigments absorb light and pass the energy from the light to the chl a in the reaction center Other accessory pigments can be present such as xanthophylls and the more well known carotenoids. The most well known carotenoid is beta-carotene which absorbs different wavelengths than the chlorophylls.

Photosystems Within the thylakoid membranes of the chloroplast, are two photosystems. Photosystem I optimally absorbs photons of a wavelength of 700 nm. Photosystem II optimally absorbs photons of a wavelength of 680 nm. Photosystem II uses light energy to oxidize two molecules of water into one molecule of molecular oxygen. The 4 electrons removed from the water molecules are transferred by an electron transport chain to ultimately reduce 2NADP+ to 2NADPH. During the electron transport process a proton gradient is generated across the thylakoid membrane. This proton motive force is then used to drive the synthesis of ATP. This process requires PSI, PSII, cytochrome bf, ferredoxin-NADP+ reductase and chloroplast ATP synthase.

Chlorophyll A and B absorb light mostly in the red and blue regions of the spectrum Carotene and xanthophyll absorb light from other regions and pass the energy to chlorophyll

Metalloenzymes

Introduction Metals play roles in approximately one-third of the known enzymes. Metals may be a co-factor or they may be incorporated into the molecule, and these are known as metalloenzymes. Amino Acids in peptide linkage posses groups that can form coordinate-covalent bonds with the metal atom. The free amino and carboxy group bind to the metal affecting the enzymes structure resulting in its active conformation (2). Metals main function is to serve in electron transfer. Many enzymes can serve as electrophiles and some can serve as nucleophilic groups. This versatility explains metals frequent occurrence in enzymes. Some metalloenzymes include hemoglobins, cytochromes, phosphotransferases, alcohol dehydrogenase, arginase, ferredoxin, and cytochrome oxidase. Carboxypeptidase A is a zinc metalloenzyme that breaks peptide linkages in the digestion of proteins. The zinc ion that the enzyme contains in its active site plays a key role in that function. Metalloenzymes can be regulated in several ways since they are such a diverse group. One way metalloenzymes are regulated is the pH level. The pH level can disrupt the electron flow that the metal would normally help facilitate. In this way the pH level could inhibit the overall effectiveness of the metalloenzyme. Transition state analogs play a key role in the competitive inhibition of metalloenzymes because they mimic the structure of the substrates transition state in the reaction of enzyme and substrate. Metalloenzymes such as the ones containing zinc can also be regulated by diet. The source of zinc in humans is almost entirely through diet. Without proper intake of metals such as zinc in a persons diet, the activity of the enzyme would be inhibited. One thing to keep in mind while studying metalloenzymes is that they are incredibly diverse and function in a multitude of important physiological processes

Structure and Overview Metalloenzymes are proteins which function as an enzyme and contain metals that are tightly bound and always isolated with the protein. In proteins such as hemoglobins and cytochromes, the metal is Fe2+ or Fe3+, and it is part of the heme prosthetic group. In other metalloenzymes the metal is built into the structure of the enzyme molecule. The metal ion can not be removed with out destroying the structure of the enzyme. Metals built into the molecule include: most phosphotransferases, containing Mg2+; alcohol dehydrogenase, Zn2+; arginase, Mn2+; ferredoxin, Fe2+; and cytochrome oxidase, Cu2+ (9). Metals are usually found in the active site of the enzyme. The metals resemble protons (H+) in that they are electrophiles that are able to accept an electron pair to form a chemical bond. In this aspect, metals may act as general acids to react with anionic and neutral ligands (2). Metal's larger size relative to protons is compensated for by their ability to react with more than one ligand. Metals typically react with two, four, or six ligands. A ligand is whatever molecule the metal interacts with. If a metal is bound with two ligands it will form a linear complex. If the metal reacts with four ligands the metal will be set in the center of a square that is planer or it will form a tetrahedral structure, and when six ligands react, the metal sits in the center of an octahedron. By clicking the following image one can view a planar arrangement of and iron- porphyrin system:

Amino acids in their peptide linkage in proteins possess groups with the ability to bind to the metal resulting in coordinate-covalent bonds. The free amino and carboxyl groups in a protein can bind to the metal and this may bind the protein to a specific, active conformation (3). The fact that metals bind to several ligands is important in that metals play a role in bringing remote parts of the amino acid sequence together and help establish an active conformation of the enzyme. Zinc is the metal incorporated in carboxypeptidase A. The zinc atom serves as a metal ion catalyst and promotes hydrolysis. The substrate fits into the hydrophobic pocket in carboxypeptidase A and zinc binds to the carboxyl group of the substrate to help stabilize the enzyme-substrate complex. In this example the zinc ion acts a generalized acid and stabilizes the developing O- as water attacks the carbonyl. Zinc can also perform a different role in enzymes like the role it performs in carbonic anhydrase. Here the metal binds H2O and makes it acidic enough to lose a proton and form a Zn-OH group. The zinc metal serves as a nucleophile to the substrate. Since zinc has the ability to act as an electrophile or as the source of a nucleophilic group it is incorporated and used by many enzymes (10).

GENERAL FUNCTIONS OF METALLOENZYMES Hemoglobins A four-subunit molecule, containing a iron atom in each subunit, in which each subunit binds a single molecule of oxygen. Hemoglobin transports oxygen from the lungs to the capillaries of the tissue. Cytochromes Cytochromes are integral membrane proteins. Cytochromes contain iron which serves to carry electrons between two segments of the electron-transport chain. The iron is reversibly oxidizable and serves as the actual electron acceptor for the cytochrome. Phosphotransferase The Mg2+ atom serves again in electron transfer. Alcohol Dehydrogenase A zinc metalloenzyme with broad specificity. They oxidize a range of aliphatic and aromatic alcohols to their corresponding aldehydes and ketones using NAD+ as a coenzyme. Arginase The metal atom of Mn2+ is used in electron transfer. Ferredoxin An electron transferring proteins involved in one-electron transfer processes. Cytochrome Oxidase The copper ions easily accommodate electron removed from a substrate and can just as easily transfer them to a molecule of oxygen (10).

Regulation and Control Metalloenzyme Inhibition Approximately one-third of the known enzymes have metals as part of their structure, require that metals be added for activity, or are further activated by metals. In enzymes where a metal has been built into the structure of the enzyme molecule, the metal cannot be removed without destroying that structure. Such enzymes include the metalloflavoproteins, the cytochromes, and the ferredoxins. In enzymes where metals are required to be added for activity the metals react reversibly with proteins to form metal-protein complexes that constitute the active catalyst. In many instances, the complex represents a specific, catalytically active conformation of the protein; the role of the metal appears to be one of stabilizing that conformation (2). Because the grouping, metalloenzymes, is so large and broad it would be almost impossible to explain how all of them can be controlled and regulated. In light of this, it is important to instead mention how the important functions of metals in enzymes can be disrupted and thus inhibited. Metals resemble protons (H+) in that they are electrophiles that are capable of accepting an electron pair to form a chemical bond. In doing so, metals may act as general acids to react with anionic and neutral ligands. This characteristic of metals is helpful in enzymatic structure and function but makes the enzyme it is part of pH dependent. Changes in pH can disrupt this electron flow that the metal would normally help facilitate and thus inhibit the overall effectiveness of the metalloenzyme. Also, because of the variability inherent to the metal's ability to react with more than one ligand, you see metals as part of the active site in many metalloenzymes. Competitive inhibitors in the form of transition-state analogs are compounds believed to look like the substrate in its transition state. To be effective the transition-state analog must not be susceptible to reaction by the enzyme. Competitive inhibition, via transition-state analog, has been exhibited in the reaction of Carboxypeptidase A by a phosphorus molecule constructed by Paul Bartlett, which can be seen in Figure 3 (10). It functions will in inhibition of CPA because the phosphorus atom, with its attached oxygens and nitrogen, resembles the tetrahedral carbon atoms in the two intermediates of Figure 2, and the transition states of all three steps (10).

Regulation of Metalloenzymes Through Diet As aforementioned, metals play a large role in the activity of a multitude of biological molecules. The source of zinc in humans is almost entirely through diet and without the intake of metals such as zinc in the diet one almost certainly inhibits the production and/or activity of many vital enzymes. Among enzymes that would not be produced by the body were it not for the presence of zinc in the body are carbonic anhydrase, the carboxypeptidases, alkaline phosphatase, lactic acid, and alcohol dehydrogenases. The recommended daily dietary allowance for zinc is 15 mg., with 20 and 25 mg. During pregnancy and lactation. The average adult human being ingests 12 to 20 mg. Of zinc per day. Deficiencies in dietary zinc intake can result in stunted growth, enlarged liver and spleen, and underdevelopment of genitals and secondary sex characteristics. Outside of dietary intake deficiencies in zinc, and thusly in enzymes that contain zinc, can be caused by the excretion of zinc in perspiration, or by blood loss if there is parasite infection. There is also increasing evidence that zinc plays an important role in protein biosynthesis and utilization. The addition of small amounts of zinc to a diet containing suboptimal amounts of a vegetable protein, as indicated by the growth of young rats, causes a pronounced increase in protein utilization and growth. This defect may result from a failure in adequate RNA synthesis. Zinc apparently inhibits the enzyme ribonuclease. Thus, in zinc deficiency, excessive destruction of RNA could occur. This demonstrates that the dietary intake of metal is not only important for the production of key enzymes but also for the inhibition of many others.

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Nitrogen Fixation. BiologicalSymbiotic Non Symbiotic AbiologicalIndustrialNatural