Article What Is the True Nitrogenase Reaction? A Guided Approach
Piero L. Ipata* Rossana Pesi
From the Department of Biology, Unit of Biochemistry, University of Pisa, 56127 Pisa, Italy
Abstract Only diazotrophic bacteria, called Rizhobia, living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N2. In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an extremely high activation energy due to the stable (unreactive) NBN triple bond. The structural and functional features of the nitrogenase protein complex, based on the stepwise transfer of eight electrons from reduced ferredoxin to the nitrogenase, coupled to the
hydrolysis of 16 ATP molecules, to fix one N2 molecule into two NH3 molecules, is well understood. Yet, a number of different nitrogenase-catalyzed reactions are present in biochemistry textbooks, which might cause misinterpretation. In this article, we show that when trying to balance the reaction catalyzed by the nitrogenase protein complex, it is important to show explicitly the 16 H1 released by the hydrolysis of the 16 ATP molecules needed to fix the C 2015 by the International Union of Bioatmospheric N2 V chemistry and Molecular Biology, 00(0):0000, 2015.
Keywords: N2 fixation; nitrogenase; balancing equation; proton omission; biochemistry teaching
Introduction Over the past three decades, the remarkable advances of molecular biology and molecular genetics have somewhat eclipsed areas of traditional biochemistry, such as enzymology and metabolism. This led to still recurring errors in the literature, although in the recent years, metabolism has reemerged as a central topic in biology [1–5]. For instance, balancing the familiar ATP dephosphorylation reaction at physiological pH (ca. 7.4) 1 ATP42 1 H2 O ! ADP32 1 P22 i 1 H
as well as the ADP phosphorylation reaction,
Moreover, students should be aware that when there is proton release or consumption, H1 and H2O may be omitted only when dealing with thermodynamic parameters, such as ’ values of ATP hydrolysis and ATP phosphorylaDGo0 and Keq tion, as both the [H2O] (55.5 M) and the [H1] (corresponding to a physiological pH of ca. 7.4) are essentially constant (see ref. [6] for a discussion on this important issue). In a biochemistry textbook [7], glycolysis is introduced as follows. The exergonic conversion of glucose into two lactate molecules and two protons C6 H12 O6 ! 2 CH3 2CHOH2COO2 1 2 H1
is added to the endergonic phosphorylation of two ADP molecules and is written as
42 1 ADP32 1 P22 i 1 H ! ATP 1 H2 O
protons are often ignored, causing incorrect interpretations even of fundamental metabolic pathways.
*Address for correspondence to: Department of Biology, Unit of Biochemistry, University of Pisa, Via San Zeno 51, 56127 Pisa, Italy. E-mail: [email protected] Received 7 October 2014; Accepted 28 November 2014 DOI 10.1002/bmb.20843 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
(1)
2 ADP 1 2 Pi ! 2 ATP 1 2 H2 O
(2)
42 1 2 ADP32 1 2 P22 1 2 H2 O i 1 2 H ! 2 ATP
(3)
instead of
By summarizing reactions (1) and (2), the following equation of oxygen-independent glycolysis is obtained: C6 H12 O6 1 2 ADP32 1 2 P22 ! 2 ATP42 i 1 2 CH3 2CHOH2COO12 1 2 H2 O 1 2 H1
(4)
It is evident that the two protons should be omitted (an excess of two positive charges are present in the right
1
Biochemistry and Molecular Biology Education side). In fact, it is well known that the glycolytic pathway per se, either starting from glucose [3] or from glycogen [4], is not an acidifying process. Protons are generated (acidosis) when the ATP molecules formed during glycolysis are hydrolyzed, not by glycolysis. Lactic acid as such is not involved. The overall oxygen-independent glycolytic equation would have been correctly written C6 H12 O6 1 2 ADP
32
1 2
P22 i
1 16 ATP42 1 16 H2 O ! 16 ADP32 1 16 P22 i 1 16 H
12
1 2 H2 O
(9)
By summarizing reactions (8) and (9), we obtain the overall reaction catalyzed by the nitrogenase system: N2 1 8 e2 1 8 H1 1 16 ATP42 1 16 H2 O ! 16 ADP32
42
! 2 ATP
1 2 CH3 2CHOH2COO
the stoichiometry of two ATPs for each electron (16 equivalents of ATP per one equivalent of N2):
1 1 2 NH3 1 H2 1 16 P22 i 1 16 H
(5)
(10)
simply if reaction (1) had been added to reaction (3) instead of reaction (2), thus avoiding disturbing misinterpretations.
The Nitrogenase-Catalyzed Reaction Ignoring H1 production by ATP hydrolysis at physiological pH may cause other ambiguities. We will focus on the conversion of atmospheric N2 into ammonia in diazotrophic microorganisms catalyzed by the nitrogenase protein complex [8]. Ammonia is then converted into ammonium ion, a useful biological form, which is assimilated into organic compounds of eukaryotic organisms. Thus, we can safely state that the nitrogen of our amino acids, purines, pyrimidines, and many other biomolecules ultimately derives from atmospheric N2. It is curious that despite the paramount importance of nitrogen fixation and the well-understood structural and functional features of the components of the metalloenzyme nitrogenase complex (see ref. [9] for a review), different overall nitrogen-catalyzed reactions are found in textbooks. The following is a discussion on the pitfalls that may cause such discrepancies. N2 fixation into two NH3 molecules requires six electrons and six protons
A cursory check showed that only in one textbook [10], reaction (10) is considered as the overall nitrogenasecatalyzed reaction. In other textbooks, protons are omitted in the right side, resulting in reactions unbalanced for charges [11–14]; see refs. [7, 13], and [14] for reaction (11) and refs. [11] and [12] for reaction (12)]: N2 1 8 e2 1 8 H1 1 16 ATP42 1 16 H2 O ! 16 ADP32 1 2 NH3 1 H2 1 16 P22 i (11) or 32 N2 1 8 e2 1 16 ATP42 1 10 H1 ! 2 NH1 4 1 16 ADP
1 16 P22 i 1 H2 (12) Omission of the 16 protons has been kept for years in the reviews written by experts in the field, most likely with the aim to show only that ATP hydrolysis occurs. It is likely that this error has been propagated in textbooks. However, there is no doubt that at least the more conscientious students when faced for the first time with the nitrogenase fixation process find it difficult to rationalize why protons are omitted in the nitrogenase reactions.
Conclusions However, the nitrogenase complex catalyzes the additional obligatory production of one hydrogen molecule: 2 H1 1 2 e2 ! H2
(7)
Therefore, eight electrons and eight protons are required to reduce one equivalent of N2 into two equivalents of ammonia and one equivalent of H2: N2 1 8 e2 18 H1 ! 2 NH3 1 H2
(8)
To “pump” the eight electrons from reduced ferredoxin (the first component of the nitrogenase system) to nitrogenase reductase (the second component), and finally to the nitrogenase enzyme protein, ATP is hydrolyzed to ADP with
2
Once produced by nitrogenase, the two ammonia molecules are almost quantitatively protonated to give two ammonium ions, because of the alkaline pKa value of ammonia: 2 H1 1 2 NH3 ! 2 NH1 4 By adding this noncatalyzed reaction to reaction (10), we obtain N2 1 8 e2 1 16 ATP42 1 10 H1 1 16 H2 O 1 ! 2 NH4 1 1 16 ADP32 1 16 P22 i 1 H2 1 16 H
(13)
This reaction should be considered as the whole process which converts atmospheric N2 into a biological useful form in all living organisms. Our discussion on the nitrogenase-catalyzed reaction suggests that there is a critical need to make it clear to students that when writing an enzyme-catalyzed reaction or
Nitrogenase Reaction
the overall equation of a metabolic pathway, we want to account for all atoms and charges in order to obey the Lavoisier’s principle. Insisting on thermodynamics may create as many problems as it solves. Finally, recalling that ATP-Mg22, rather than ATP42 [15], is one of the substrates, the reaction (13) becomes N2 1 8 e2 1 16 ATP2Mg22 1 10 H1 1 16 H2 O 12 22 1 ! 2 NH1 4 1 16 ADP2Mg 1 16 Pi 1 H2 1 16 H
(14)
Acknowledgement This study was supported by local funds of the University of Pisa, Italy.
REFERENCES [1] Bar-Even, A., Flamholz, A., Noor, E., and Milo, R. (2012) Rethinking glycolysis: On the biochemical logic of metabolic pathways. Nat. Chem. Biol. 8, 509–517. [2] Lane, A. N., Fan, T. W. M., and Higashi, R. M. (2009) Metabolic acidosis and the importance of balanced equations. Metabolomics 5, 163–165. [3] Robergs, R. A., Ghiasvand, F., and Parker, D. (2004) Biochemistry of exercise induced metabolic acidosis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R502–R516.
Ipata and Pesi
[4] Ipata, P. L. and Balestri, F. (2012) Glycogen as a fuel: Metabolic interaction between glycogen and ATP catabolism in oxygen-independent muscle contraction. Metabolomics 8, 736–741. [5] Robergs, R. A., Ghiasvand, F., and Parker, D. (2005) Lingering construct of lactic acidosis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, R904–R910. [6] Alberty, R. A., Cornish-Bowden, A., Goldberg, R. N., Hammes, G. G., Tipton, K., and Westerhoff, H. V. (2011) Recommendations for terminology and databases for biochemical thermodynamics. Biophys. Chem. 155, 89–103. [7] Garrett, R. H. and Grisham, C. M. (1999) Biochemistry, 2nd ed., Thomson, Brooks/Cole, PACIFIC GROVE, CA, 93950, USA. [8] Kim, J. and Rees, D. C. (1989) Nitrogenase and biological nitrogen fixation. Biochemistry 33, 389–397. [9] Hu, Y. and Ribbe, M. W. (2013) Nitrogenase assembly. Biochim. Biophys Acta 1827, 1112–1122. [10] Berg, J. M., Tymoczko, J. L., and Stryer, L. (2012) Biochemistry, 7th ed., W. H. Freeman and Co., New York. [11] Nelson, D. L. and Cox, M. (2008) Lehninger Principles of Biochemistry, 4th ed., John Wiley and sons, New York. [12] Campbell, M. K. and Farrell, S. O. (2012) Biochemistry, 7th ed., Thomson, Brooks/Cole, PACIFIC GROVE, CA, 93950, USA. [13] Voet, D., Voet, J., and Pratt, C. W. (2013) Fundamental of Biochemistry, 4th ed., John Wiley and sons, New York. [14] Moran, L. A., Scrimgeour, K. G., Horton, H. R., Ochs, R. S., and Rawn, J. D. (1996) Biochemistry, 2nd ed., Mc Graw-Hill, New York. [15] Storer, A. C. and Cornish-Bowden, A. (1976) Concentration of MgATP22 and other ions in solution. Calculation of the true concentration of species present in mixtures of associating ions. Biochem. J. 159, 1–5.
3
Piero L. Ipata* Rossana Pesi
From the Department of Biology, Unit of Biochemistry, University of Pisa, 56127 Pisa, Italy
Abstract Only diazotrophic bacteria, called Rizhobia, living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N2. In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an extremely high activation energy due to the stable (unreactive) NBN triple bond. The structural and functional features of the nitrogenase protein complex, based on the stepwise transfer of eight electrons from reduced ferredoxin to the nitrogenase, coupled to the
hydrolysis of 16 ATP molecules, to fix one N2 molecule into two NH3 molecules, is well understood. Yet, a number of different nitrogenase-catalyzed reactions are present in biochemistry textbooks, which might cause misinterpretation. In this article, we show that when trying to balance the reaction catalyzed by the nitrogenase protein complex, it is important to show explicitly the 16 H1 released by the hydrolysis of the 16 ATP molecules needed to fix the C 2015 by the International Union of Bioatmospheric N2 V chemistry and Molecular Biology, 00(0):0000, 2015.
Keywords: N2 fixation; nitrogenase; balancing equation; proton omission; biochemistry teaching
Introduction Over the past three decades, the remarkable advances of molecular biology and molecular genetics have somewhat eclipsed areas of traditional biochemistry, such as enzymology and metabolism. This led to still recurring errors in the literature, although in the recent years, metabolism has reemerged as a central topic in biology [1–5]. For instance, balancing the familiar ATP dephosphorylation reaction at physiological pH (ca. 7.4) 1 ATP42 1 H2 O ! ADP32 1 P22 i 1 H
as well as the ADP phosphorylation reaction,
Moreover, students should be aware that when there is proton release or consumption, H1 and H2O may be omitted only when dealing with thermodynamic parameters, such as ’ values of ATP hydrolysis and ATP phosphorylaDGo0 and Keq tion, as both the [H2O] (55.5 M) and the [H1] (corresponding to a physiological pH of ca. 7.4) are essentially constant (see ref. [6] for a discussion on this important issue). In a biochemistry textbook [7], glycolysis is introduced as follows. The exergonic conversion of glucose into two lactate molecules and two protons C6 H12 O6 ! 2 CH3 2CHOH2COO2 1 2 H1
is added to the endergonic phosphorylation of two ADP molecules and is written as
42 1 ADP32 1 P22 i 1 H ! ATP 1 H2 O
protons are often ignored, causing incorrect interpretations even of fundamental metabolic pathways.
*Address for correspondence to: Department of Biology, Unit of Biochemistry, University of Pisa, Via San Zeno 51, 56127 Pisa, Italy. E-mail: [email protected] Received 7 October 2014; Accepted 28 November 2014 DOI 10.1002/bmb.20843 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
(1)
2 ADP 1 2 Pi ! 2 ATP 1 2 H2 O
(2)
42 1 2 ADP32 1 2 P22 1 2 H2 O i 1 2 H ! 2 ATP
(3)
instead of
By summarizing reactions (1) and (2), the following equation of oxygen-independent glycolysis is obtained: C6 H12 O6 1 2 ADP32 1 2 P22 ! 2 ATP42 i 1 2 CH3 2CHOH2COO12 1 2 H2 O 1 2 H1
(4)
It is evident that the two protons should be omitted (an excess of two positive charges are present in the right
1
Biochemistry and Molecular Biology Education side). In fact, it is well known that the glycolytic pathway per se, either starting from glucose [3] or from glycogen [4], is not an acidifying process. Protons are generated (acidosis) when the ATP molecules formed during glycolysis are hydrolyzed, not by glycolysis. Lactic acid as such is not involved. The overall oxygen-independent glycolytic equation would have been correctly written C6 H12 O6 1 2 ADP
32
1 2
P22 i
1 16 ATP42 1 16 H2 O ! 16 ADP32 1 16 P22 i 1 16 H
12
1 2 H2 O
(9)
By summarizing reactions (8) and (9), we obtain the overall reaction catalyzed by the nitrogenase system: N2 1 8 e2 1 8 H1 1 16 ATP42 1 16 H2 O ! 16 ADP32
42
! 2 ATP
1 2 CH3 2CHOH2COO
the stoichiometry of two ATPs for each electron (16 equivalents of ATP per one equivalent of N2):
1 1 2 NH3 1 H2 1 16 P22 i 1 16 H
(5)
(10)
simply if reaction (1) had been added to reaction (3) instead of reaction (2), thus avoiding disturbing misinterpretations.
The Nitrogenase-Catalyzed Reaction Ignoring H1 production by ATP hydrolysis at physiological pH may cause other ambiguities. We will focus on the conversion of atmospheric N2 into ammonia in diazotrophic microorganisms catalyzed by the nitrogenase protein complex [8]. Ammonia is then converted into ammonium ion, a useful biological form, which is assimilated into organic compounds of eukaryotic organisms. Thus, we can safely state that the nitrogen of our amino acids, purines, pyrimidines, and many other biomolecules ultimately derives from atmospheric N2. It is curious that despite the paramount importance of nitrogen fixation and the well-understood structural and functional features of the components of the metalloenzyme nitrogenase complex (see ref. [9] for a review), different overall nitrogen-catalyzed reactions are found in textbooks. The following is a discussion on the pitfalls that may cause such discrepancies. N2 fixation into two NH3 molecules requires six electrons and six protons
A cursory check showed that only in one textbook [10], reaction (10) is considered as the overall nitrogenasecatalyzed reaction. In other textbooks, protons are omitted in the right side, resulting in reactions unbalanced for charges [11–14]; see refs. [7, 13], and [14] for reaction (11) and refs. [11] and [12] for reaction (12)]: N2 1 8 e2 1 8 H1 1 16 ATP42 1 16 H2 O ! 16 ADP32 1 2 NH3 1 H2 1 16 P22 i (11) or 32 N2 1 8 e2 1 16 ATP42 1 10 H1 ! 2 NH1 4 1 16 ADP
1 16 P22 i 1 H2 (12) Omission of the 16 protons has been kept for years in the reviews written by experts in the field, most likely with the aim to show only that ATP hydrolysis occurs. It is likely that this error has been propagated in textbooks. However, there is no doubt that at least the more conscientious students when faced for the first time with the nitrogenase fixation process find it difficult to rationalize why protons are omitted in the nitrogenase reactions.
Conclusions However, the nitrogenase complex catalyzes the additional obligatory production of one hydrogen molecule: 2 H1 1 2 e2 ! H2
(7)
Therefore, eight electrons and eight protons are required to reduce one equivalent of N2 into two equivalents of ammonia and one equivalent of H2: N2 1 8 e2 18 H1 ! 2 NH3 1 H2
(8)
To “pump” the eight electrons from reduced ferredoxin (the first component of the nitrogenase system) to nitrogenase reductase (the second component), and finally to the nitrogenase enzyme protein, ATP is hydrolyzed to ADP with
2
Once produced by nitrogenase, the two ammonia molecules are almost quantitatively protonated to give two ammonium ions, because of the alkaline pKa value of ammonia: 2 H1 1 2 NH3 ! 2 NH1 4 By adding this noncatalyzed reaction to reaction (10), we obtain N2 1 8 e2 1 16 ATP42 1 10 H1 1 16 H2 O 1 ! 2 NH4 1 1 16 ADP32 1 16 P22 i 1 H2 1 16 H
(13)
This reaction should be considered as the whole process which converts atmospheric N2 into a biological useful form in all living organisms. Our discussion on the nitrogenase-catalyzed reaction suggests that there is a critical need to make it clear to students that when writing an enzyme-catalyzed reaction or
Nitrogenase Reaction
the overall equation of a metabolic pathway, we want to account for all atoms and charges in order to obey the Lavoisier’s principle. Insisting on thermodynamics may create as many problems as it solves. Finally, recalling that ATP-Mg22, rather than ATP42 [15], is one of the substrates, the reaction (13) becomes N2 1 8 e2 1 16 ATP2Mg22 1 10 H1 1 16 H2 O 12 22 1 ! 2 NH1 4 1 16 ADP2Mg 1 16 Pi 1 H2 1 16 H
(14)
Acknowledgement This study was supported by local funds of the University of Pisa, Italy.
REFERENCES [1] Bar-Even, A., Flamholz, A., Noor, E., and Milo, R. (2012) Rethinking glycolysis: On the biochemical logic of metabolic pathways. Nat. Chem. Biol. 8, 509–517. [2] Lane, A. N., Fan, T. W. M., and Higashi, R. M. (2009) Metabolic acidosis and the importance of balanced equations. Metabolomics 5, 163–165. [3] Robergs, R. A., Ghiasvand, F., and Parker, D. (2004) Biochemistry of exercise induced metabolic acidosis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R502–R516.
Ipata and Pesi
[4] Ipata, P. L. and Balestri, F. (2012) Glycogen as a fuel: Metabolic interaction between glycogen and ATP catabolism in oxygen-independent muscle contraction. Metabolomics 8, 736–741. [5] Robergs, R. A., Ghiasvand, F., and Parker, D. (2005) Lingering construct of lactic acidosis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, R904–R910. [6] Alberty, R. A., Cornish-Bowden, A., Goldberg, R. N., Hammes, G. G., Tipton, K., and Westerhoff, H. V. (2011) Recommendations for terminology and databases for biochemical thermodynamics. Biophys. Chem. 155, 89–103. [7] Garrett, R. H. and Grisham, C. M. (1999) Biochemistry, 2nd ed., Thomson, Brooks/Cole, PACIFIC GROVE, CA, 93950, USA. [8] Kim, J. and Rees, D. C. (1989) Nitrogenase and biological nitrogen fixation. Biochemistry 33, 389–397. [9] Hu, Y. and Ribbe, M. W. (2013) Nitrogenase assembly. Biochim. Biophys Acta 1827, 1112–1122. [10] Berg, J. M., Tymoczko, J. L., and Stryer, L. (2012) Biochemistry, 7th ed., W. H. Freeman and Co., New York. [11] Nelson, D. L. and Cox, M. (2008) Lehninger Principles of Biochemistry, 4th ed., John Wiley and sons, New York. [12] Campbell, M. K. and Farrell, S. O. (2012) Biochemistry, 7th ed., Thomson, Brooks/Cole, PACIFIC GROVE, CA, 93950, USA. [13] Voet, D., Voet, J., and Pratt, C. W. (2013) Fundamental of Biochemistry, 4th ed., John Wiley and sons, New York. [14] Moran, L. A., Scrimgeour, K. G., Horton, H. R., Ochs, R. S., and Rawn, J. D. (1996) Biochemistry, 2nd ed., Mc Graw-Hill, New York. [15] Storer, A. C. and Cornish-Bowden, A. (1976) Concentration of MgATP22 and other ions in solution. Calculation of the true concentration of species present in mixtures of associating ions. Biochem. J. 159, 1–5.
3
