Editing Leghemoglobin

{{Short description|Oxygen-carrying phytoglobin found in rhizome of leguminous plants}}
[[File:Leghemoglobin A.png|thumb|upright=1.35|Leghemoglobin A from a soybean (PDB: 1BIN)]]
{{Infobox protein family
|Name=Leghaemoglobin, iron-binding site
|Symbol=Leghaemoglobin_Fe_BS
|PROSITE=PS00208
|InterPro=IPR019824
}}

'''Leghemoglobin''' (also '''leghaemoglobin''' or '''legoglobin''') is an [[Oxygen-carrying protein|oxygen-carrying]] [[phytoglobin]] found in the [[Nitrogen fixation|nitrogen-fixing]] [[root nodule]]s of [[legume|leguminous]] plants. It is produced by these plants in response to the roots being colonized by nitrogen-fixing bacteria, termed [[rhizobia]], as part of the [[symbiosis|symbiotic]] interaction between plant and bacterium: roots not colonized by ''[[Rhizobium]]'' do not synthesise leghemoglobin. Leghemoglobin has close chemical and structural similarities to [[hemoglobin]], and, like hemoglobin, is red in colour. It was originally thought that the [[heme]] prosthetic group for plant leghemoglobin was provided by the bacterial symbiont within symbiotic root nodules.<ref>{{cite journal |vauthors=Nadler KD, Avissar YJ |title=Heme Synthesis in Soybean Root Nodules: I. On the Role of Bacteroid delta-Aminolevulinic Acid Synthase and delta-Aminolevulinic Acid Dehydrase in the Synthesis of the Heme of Leghemoglobin |journal=Plant Physiology |volume=60 |issue=3 |pages=433–6 |date=September 1977 |pmid=16660108 |pmc=542631 |doi=10.1104/pp.60.3.433}}</ref><ref>{{cite journal |vauthors=O'Brian MR, Kirshbom PM, Maier RJ |title=Bacterial heme synthesis is required for expression of the leghemoglobin holoprotein but not the apoprotein in soybean root nodules |journal=Proceedings of the National Academy of Sciences of the United States of America |volume=84 |issue=23 |pages=8390–3 |date=December 1987 |pmid=3479799 |pmc=299548 |doi=10.1073/pnas.84.23.8390 |bibcode=1987PNAS...84.8390O |doi-access=free}}</ref> However, subsequent work shows that the plant host strongly expresses heme biosynthesis genes within nodules, and that activation of those genes correlates with leghemoglobin gene expression in developing nodules.<ref>{{cite journal |vauthors=Sangwan I, O'Brian MR |title=Evidence for an inter-organismic heme biosynthetic pathway in symbiotic soybean root nodules |journal=Science |volume=251 |issue=4998 |pages=1220–2 |date=March 1991 |pmid=17799282 |doi=10.1126/science.251.4998.1220 |s2cid=11471787 |bibcode=1991Sci...251.1220S}}</ref><ref>{{cite journal |vauthors=Sangwan I, O'Brian MR |title=Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules |journal=Plant Physiology |volume=98 |issue=3 |pages=1074–9 |date=March 1992 |pmid=16668729 |pmc=1080310 |doi=10.1104/pp.98.3.1074}}</ref><ref>{{cite journal |vauthors=Sangwan I, O'Brian MR |title=Expression of the soybean (Glycine max) glutamate 1-semialdehyde aminotransferase gene in symbiotic root nodules |journal=Plant Physiology |volume=102 |issue=3 |pages=829–34 |date=July 1993 |pmid=8278535 |pmc=158853 |doi=10.1104/pp.102.3.829}}</ref><ref>{{cite journal |vauthors=Madsen O, Sandal L, Sandal NN, Marcker KA |title=A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules |journal=Plant Molecular Biology |volume=23 |issue=1 |pages=35–43 |date=October 1993 |pmid=8219054 |doi=10.1007/BF00021417 |s2cid=23011457}}</ref><ref>{{cite journal |vauthors=Kaczor CM, Smith MW, Sangwan I, O'Brian MR |title=Plant delta-aminolevulinic acid dehydratase. Expression in soybean root nodules and evidence for a bacterial lineage of the Alad gene |journal=Plant Physiology |volume=104 |issue=4 |pages=1411–7 |date=April 1994 |pmid=8016269 |pmc=159307 |doi=10.1104/pp.104.4.1411}}</ref><ref>{{cite journal |vauthors=Frustaci JM, Sangwan I, O'Brian MR |title=gsa1 is a universal tetrapyrrole synthesis gene in soybean and is regulated by a GAGA element |journal=The Journal of Biological Chemistry |volume=270 |issue=13 |pages=7387–93 |date=March 1995 |pmid=7706283 |doi=10.1074/jbc.270.13.7387 |doi-access=free}}</ref><ref>{{cite journal |vauthors=Santana MA, Pihakaski-Maunsbach K, Sandal N, Marcker KA, Smith AG |title=Evidence that the plant host synthesizes the heme moiety of leghemoglobin in root nodules |journal=Plant Physiology |volume=116 |issue=4 |pages=1259–69 |date=April 1998 |pmid=9536042 |pmc=35032 |doi=10.1104/pp.116.4.1259}}</ref><ref>{{cite journal |vauthors=Sangwan I, O'Brian MR |title=Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules |journal=Plant Physiology |volume=119 |issue=2 |pages=593–8 |date=February 1999 |pmid=9952455 |pmc=32136 |doi=10.1104/pp.119.2.593}}</ref>

In plants colonised by ''Rhizobium'', such as [[alfalfa]] or [[soybean]]s, the presence of [[oxygen]] in the root nodules would reduce the activity of the oxygen-sensitive [[nitrogenase]], which is an enzyme responsible for the fixation of atmospheric nitrogen. Leghemoglobin is shown to buffer the concentration of free oxygen in the [[cytoplasm]] of infected plant cells to ensure the proper function of root nodules. That being said, nitrogen fixation is an extremely energetically costly process, so [[aerobic respiration]], which necessitates high oxygen concentration, is necessary in the cells of the root nodule.<ref>{{cite book |last1=Berg |first1=J. |last2=Tymoczko |first2=J. |last3=Gatto Jr. |first3=G. |last4=Stryer |first4=L. |title=Biochemistry |edition=8th |publisher=W.H. & Freeman Company |year=2015}}</ref> Leghemoglobin maintains a free oxygen concentration that is low enough to allow nitrogenase to function, but a high enough total oxygen concentration (free and bound to leghemoglobin) for aerobic respiration.

Leghemoglobin falls into the class of '''symbiotic globins''', which also include the root nodules globins of [[actinorhizal plant]]s such as ''[[Casuarina]]''. The ''Casuarina'' symbiotic globin is intermediate between leghemoglobin and nonsymbiotic phytoglobin-2.<ref name="phytoglobin" /><ref name="Hill16">{{cite journal |vauthors=Hill R, Hargrove MS, Arredondo-Peter R |title=Phytoglobin: a novel nomenclature for plant globins accepted by the globin community at the 2014 XVIII conference on Oxygen-Binding and Sensing Proteins |journal=F1000Research |date=2016 |volume=5 |page=212 |doi=10.12688/f1000research.8133.1 |pmid=26998237 |pmc=4792203 |doi-access=free }}</ref>

== Structure ==

Leghemoglobins are monomeric proteins with a mass around 16 kDa, and are structurally similar to [[myoglobin]].<ref name = Singh>Singh S., Varma A. (2017) Structure, Function, and Estimation of Leghemoglobin. In: Hansen A., Choudhary D., Agrawal P., Varma A. (eds) Rhizobium Biology and Biotechnology. Soil Biology, vol 50. Springer, Cham</ref> One leghemoglobin protein consists of a heme bound to an iron, and one polypeptide chain (the globin).<ref name=Singh/> Similar to myoglobin and hemoglobin, the iron of heme is found in its [[ferrous]] state in vivo, and is the moiety that binds oxygen.<ref name=Singh/> Despite similarities in the mechanism of oxygen binding between leghemoglobin and animal hemoglobin, and the fact that leghemoglobin and animal hemoglobin evolved from a common ancestor, there is dissimilarity in amino acid sequence between these proteins at about 80% of positions.<ref name=Singh/>
[[File:Lba Oxygen Stabilization.png|thumb|379x379px|Oxygen Stabilization of Leghemoglobin A (PDB: 1BIN)]]
Oxygen [[binding affinities]] of leghemoglobins are between 11 and 24 times higher than oxygen binding affinities of sperm whale myoglobin.<ref name="ReferenceA">{{cite journal |title=The structure of deoxy- and oxy-leghaemoglobin from lupin |vauthors=Harutyunyan EH, Safonova TN, Kuranova IP, Popov AN, Teplyakov AV, Obmolova GV, Rusakov AA, Vainshtein BK, Dodson GG, Wilson JC}}{{fcn|date=May 2023}}</ref> Differences in the affinities are due to differential rates of association between the two types of proteins.<ref name="ReferenceA"/> One explanation of this phenomenon is that in myoglobin, a bound water molecule is stabilized in a pocket surrounding the heme group. This water group must be displaced in order for oxygen to bind. No such water is bound in the analogous pocket of leghemoglobin, so it is easier for an oxygen molecule to approach the leghemoglobin heme.<ref name=Singh/> Leghemoglobin has a slow oxygen dissociation rate, similar to myoglobin.<ref name=Wittenberg>{{cite journal |vauthors=Wittenberg JB, Appleby CA, Wittenberg BA |date=January 1972 |title=The Kinetics of the Reactions of Leghemoglobin with Oxygen and Carbon Monoxide |journal=Journal of Biological Chemistry |volume=247 |issue=2 |pages=527–531 |doi=10.1016/S0021-9258(19)45734-7 |pmid=4333266 |doi-access=free |url=https://www.jbc.org/article/S0021-9258(19)45734-7/pdf}}</ref> Like myoglobin and hemoglobin, leghemoglobin has a high affinity for carbon monoxide.<ref name=Wittenberg/>

In the primary structure of Leghemoglobin A in soybeans, a valine(F7) is found in place where a serine(F7) is in Myoglobin. Without a hydrogen bond fixing the orientation of the proximal histidine side chain the imidazole ring can occupy a staggered conformation between pyrrole nitrogen atoms and can readily move upward to the heme plane. This greatly increases the reactivity of the iron atom and oxygen affinity. In Leghemoglobin A the distal histidine side chain is also rotated away from the bound ligand by formation of a hydrogen bond with Tyrosine.<ref>{{Cite journal |last1=Smagghe |first1=Benoit J. |last2=Hoy |first2=Julie A. |last3=Percifield |first3=Ryan |last4=Kundu |first4=Suman |last5=Hargrove |first5=Mark S. |last6=Sarath |first6=Gautam |last7=Hilbert |first7=Jean-Louis |last8=Watts |first8=Richard A. |last9=Dennis |first9=Elizabeth S. |last10=Peacock |first10=W. James |last11=Dewilde |first11=Sylvia |last12=Moens |first12=Luc |last13=Blouin |first13=George C. |last14=Olson |first14=John S. |last15=Appleby |first15=Cyril A. |date=December 2009 |title=Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins |journal=Biopolymers |volume=91 |issue=12 |pages=1083–1096 |doi=10.1002/bip.21256 |issn=0006-3525 |pmid=19441024|s2cid=1891302 }}</ref>

Heme groups are the same in all known leghemoglobins, but the amino acid sequence of the globin differs slightly depending on bacterial strain and legume species.<ref name=Singh/> Even within one leguminous plant, multiple [[Protein isoform|isoforms]] of leghemoglobins can exist. These often differ in oxygen affinity, and help meet the needs of a cell in a particular environment within the nodule.<ref>{{cite journal |vauthors=Kawashima K, Suganuma N, Tamaoki M, Kouchi H |title=Two types of pea leghemoglobin genes showing different O2-binding affinities and distinct patterns of spatial expression in nodules |journal=Plant Physiol. |date=2001 |volume=125 |issue=2 |pages=641–651 |doi=10.1104/pp.125.2.641|pmid=11161022 |pmc=64866 }}</ref>

== Debate on principal function ==

Results of a 1995 study suggested that the low free oxygen concentration in root nodule cells is actually due to the low oxygen permeability of root nodule cells.<ref>{{cite book |vauthors=Ludwig RA, de Vries GE |veditors=Broughton WJ, Pühler S |title=Nitrogen Fixation, Vol. 4: Molecular Biology |chapter=Biochemical physiology of Rhizobium dinitrogen fixation |publisher=Clarendon University Press |year=1986 |location=Oxford, UK |pages=[https://archive.org/details/nitrogenfixation0000unse_h1p7/page/50 50–69] |isbn=978-0-19-854575-0 |chapter-url=https://archive.org/details/nitrogenfixation0000unse_h1p7}}</ref> It follows that the main purpose of leghemoglobin is to scavenge the limited free oxygen in the cell and deliver it to [[mitochondria]] for respiration. But, scientists of a later 2005 article suggest that leghemoglobin is responsible both for buffering oxygen concentration, and for delivery of oxygen to mitochondria.<ref name=pubmed.15797021>{{Cite journal |last1=Ott |first1=Thomas |last2=van Dongen |first2=Joost T. |last3=Günther |first3=Catrin |last4=Krusell |first4=Lene |last5=Desbrosses |first5=Guilhem |last6=Vigeolas |first6=Helene |last7=Bock |first7=Vivien |last8=Czechowski |first8=Tomasz |last9=Geigenberger |first9=Peter |last10=Udvardi |first10=Michael K. |date=2005-03-29 |title=Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development |journal=Current Biology |volume=15 |issue=6 |pages=531–535 |doi=10.1016/j.cub.2005.01.042 |issn=0960-9822 |pmid=15797021|doi-access=free |bibcode=2005CBio...15..531O }}</ref> Their leghemoglobin [[Gene knockout|knockout]] studies showed that leghemoglobin actually does significantly decrease the free oxygen concentration in root nodule cells, and that nitrogenase expression was eliminated in leghemoglobin knockout mutants, assumably due to the degradation of nitrogenase with high free oxygen concentration. Their study also showed a higher [[Adenosine triphosphate|ATP]]/[[Adenosine diphosphate|ADP]] ratio in wild-type root nodule cells with active leghemoglobin, suggesting that leghemoglobin also assists with delivery of oxygen for respiration. 

Plants contain both symbiotic and nonsymbiotic hemoglobins. Symbiotic hemoglobins are thought to be important for symbiotic nitrogen fixation (SNF). In legume, SNF takes place in specialized organs called nodules which contain bacteroids, or nitrogen fixing rhizobia. The induction of nodule-specific plant genes, which include those that encode for symbiotic leghemoglobins (Lb), accompany nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation to buffer free oxygen in the nanomolar range, which can avoid inactivation of oxygen-labile nitrogenase while keeping a high enough oxygen flux for respiration in the cell. The leghemoglobins are required for SNF but are not required for plant growth and development in the presence of an external source of fixed nitrogen. Leghemoglobins make the essential contribution of establishing low free-oxygen concentrations while keep a high energy status in cells. These are the conditions necessary for effective SNF.<ref name=pubmed.15797021/>

== Other plant hemoglobins ==
{{main|Phytoglobin}}
Globins have since been identified as a protein common to many plant taxa, not restricted to symbiotic ones. In light of this discovery, it has been proposed that the term phytoglobins be used for referring to plant globins in general.<ref name="phytoglobin">{{cite journal |last1=Becana |first1=Manuel |last2=Yruela |first2=Inmaculada |last3=Sarath |first3=Gautam |last4=Catalán |first4=Pilar |last5=Hargrove |first5=Mark S. |title=Plant hemoglobins: a journey from unicellular green algae to vascular plants |journal=New Phytologist |date=September 2020 |volume=227 |issue=6 |pages=1618–1635 |doi=10.1111/nph.16444|pmid=31960995 |doi-access=free |bibcode=2020NewPh.227.1618B |hdl=10261/219101 |hdl-access=free }}</ref>

Phytoglobins can be divided into two clades. The 3/3-fold type contains Classes I and II of angiosperm phytoglobins, and is the one common to all eukaryotes ([[horizontal gene transfer|HGT]] of a bacterial flavohemoglobin). The leghemoglobin ''sensu stricto'' is a class II phytoglobin. The 2/2-fold "TrHb2" type contains class III in angiosperm nomenclature, and appears to be acquired from [[Chloroflexota]] (formerly Chloroflexi) by the ancestor of land plants.<ref name="phytoglobin"/>

== Commercial use ==
[[Impossible Foods]] asked the American [[Food and Drug Administration|FDA]] for their approval to use recombinant soy leghemoglobin made by ''[[Pichia pastoris]]'' in [[Meat alternative|meat alternatives]] as an [[Functional analog (chemistry)|analog]] of meat-derived [[hemoglobin]].<ref>{{Cite web |title=GRAS Notice 540 |url=https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=540 |website=www.accessdata.fda.gov |access-date=2018-01-21 |archive-date=2017-06-30 |archive-url=https://web.archive.org/web/20170630052957/https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=540 |url-status=dead }}</ref><ref>{{Cite web |url=https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=737 |archive-url=https://web.archive.org/web/20180504172520/https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=737 |url-status=dead |archive-date=May 4, 2018 |title=GRAS Notice 737 |website=www.accessdata.fda.gov |access-date=2018-08-22 }}</ref> Approval from the FDA came in July 2019,<ref>{{Cite web |url=https://www.cnbc.com/2019/07/31/beyond-meats-competitor-impossible-foods-gets-fda-approval.html |title=Beyond Meat's competitor Impossible Foods plans to launch in grocery stores in September after getting FDA approval |date=31 July 2019 |website=CNBC |access-date=31 July 2019}}</ref> was challenged,{{Efn|filed by the non-profit advocacy organization Center for Food Safety}} and later upheld, on May 3, 2021, by a San Francisco federal appeals court.<ref>{{cite web |author=Justine Calma |url=https://www.theverge.com/2021/5/3/22418036/impossible-foods-legal-battle-key-ingredient-heme-fda |date=May 3, 2021 |title=Impossible Foods clears legal battle over the ingredient that makes its meat 'bleed' |website=theverge.com |url-status=live |archive-url=https://web.archive.org/web/20210507190036/https://www.theverge.com/2021/5/3/22418036/impossible-foods-legal-battle-key-ingredient-heme-fda |archive-date=2021-05-07}}</ref><ref>{{cite web |author=Sally Ho |date=May 6, 2021 |url=https://www.greenqueen.com.hk/impossible-foods-wins-legal-battle-over-heme-ingredient-powering-bleeding-plant-based-burger/ |title=Impossible Foods Wins Legal Battle Over Heme Ingredient Powering 'Bleeding' Plant-Based Burger |website=greenqueen.com.hk |url-status=live |archive-url=https://web.archive.org/web/20210506070157/https://www.greenqueen.com.hk/impossible-foods-wins-legal-battle-over-heme-ingredient-powering-bleeding-plant-based-burger/ |archive-date=2021-05-06}}</ref> It is currently being used in their products to mimic the color, taste, and texture of meat.<ref>{{cite web |last=Bandoim |first=L. |date=December 20, 2019 |title=What The FDA's Decision About Soy Leghemoglobin Means For Impossible Burger |website=[[Forbes]] |url=https://www.forbes.com/sites/lanabandoim/2019/12/20/what-the-fdas-decision-about-soy-leghemoglobin-means-for-impossible-burger/#5e0a8c7457f6 |access-date=March 4, 2020}}</ref>

== See also ==
{{Commons category}}
* [[Hemocyanin]]
* [[Hemoglobin]]
* [[Myoglobin]]
* [[Phytoglobin]]

== References ==
{{Reflist}}

== Notes ==
{{notelist}}

== Further reading ==
<!-- The following two references need to be incorporated into the text. -->
* {{cite journal |vauthors=Virtanen AI |title=Biological nitrogen fixation |journal=Annual Review of Microbiology |volume=2 |issue=1 |pages=485–506 |year=1948 |pmid=18122253 |doi=10.1146/annurev.mi.02.100148.002413}}
* {{cite book |last1=Taiz |first1=L. |last2=Zeiger |first2=E. |title=Plant Physiology Online |edition=3rd |publisher=Sinauwr Associates, Inc |year=2006 |location=Sunderland, MA |pages=269 |isbn=978-0-87893-856-8 |url=http://4e.plantphys.net/ |access-date=2008-05-03 |archive-date=2008-05-09 |archive-url=https://web.archive.org/web/20080509053904/http://4e.plantphys.net/ |url-status=dead }}
*[https://www.nytimes.com/2017/08/08/business/impossible-burger-food-meat.html? Impossible Burger’s ‘Secret Sauce’ Highlights Challenges of Food Tech]
*[https://www.fda.gov/food/cfsan-constituent-updates/fda-announces-effective-date-final-rule-adding-soy-leghemoglobin-list-color-additives-exempt Updates FDA Announces Effective Date for Final Rule Adding Soy Leghemoglobin to List of Color Additives Exempt from Certification]

{{Globins}}

[[Category:Hemoproteins]]
[[Category:Nitrogen cycle]]
[[Category:Phytochemicals]]
[[Category:Hemoglobins]]