MOLYBDATE METABOLISM OP AZOTOBACTER DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By RICHARD FAIRBANKS KEELER, B. S., M. S. The Ohio State University 1957 Approved by: Adviser Department of Agricultural Biochemistry ACKNOWLEDGMENTS The author wishes to express his appreciation to his adviser, Dr, <J. E. Varner, to Dr. G. C. Webster, and to the other members of the Department of Agricultural Biochemistry for their help and guidance during the course of his graduate program. Thanks go to L. B. Carr for the micrographs shown herein, and to Dr. H. J. Hausman and Paul Weiler for the preparation of the N*^. Financial support for this investigation was very generously provided by the Research Corporation. i± TABLE OF CONTENTS Page INTROHJCTION 1 REVIEW OF IRE LITERATURE 4 EXPERIMENTAL METHODS 16 RESULTS AND DISCUSSION 23 SUMMARY 61 CONCLUSIONS AND OUTLOOK 65 BIBLIOGRAPHY 106 AUTOBIOGRAPHY 111 iii LIST OP ILLUSTRATIONS Figure Page 1. Serial (NH4 )qS04 Fractionation of the Proteins of the Azotobacter vinelandil 25,000 Times Gravity Supernatant Fraction " 68 2. Paper Electrophoresis of Radioactive Tungsto- and Molybdoproteins from Partially Purified Homogenates of Azotobacter vinelandii Grown on N2 69 3. Serial (NH4 )gS04 Fractionation of the Molybdo­ proteins of the Azotobacter chroococcum 25,000 Times Gravity Supernatant Fraction 70 4. 1,500 Times Magnified Azotobacter vinelandii Preparations Photographed under tight Microscopy 71 5* 15,000 Times Magnified Electron Micrograph of the Membrane Preparation of Azotobacter vinelandii. 72 iv LIST OF TABLES Table Page 99 1. Uptake and Distribution of Mo in Centri­ fugal Fractions of Azotobacter vinelandii 73 2* Uptake and Distribution of M o " in Centri­ fugal Fractions of Azotobacter vinelandii 74 QQ 3. Mo Uptake by Azotobacter vinelandii as a Function of Growth 75 4. M o " Uptake by Azotobacter vinelandii as a Function of Aeration 76 99 5. Mo Uptake by Azotobacter vinelandii as a Function of the Iron Concentration 77 59 6 . Iron Uptake by Azotobacter vinelandii growing on TTg as a Function of Aeration 78 7. M o " Uptake and Distribution In Azotobacter vinelandii as a Function of Culture Con­ ditions » 79 8 . Effects of Tungsten, Vanadium, and Molybde­ num on the Growth of Azotobacter vinelandii 80 9. Tungsten as a Competitive Inhibitor of Molybde­ num in the Growth of Azotobacter vinelandii 81 10* The Effect of Tungsten on the Uptake of M o " by Azotobacter vinelandii 82 QQ 11. Uptake of Mo by Azotobacter vinelandii as a Function of the tungsten Level in the Medium 83 12. Uptake of w185 by Azotobacter vinelandii as a Function of the Molybdenum Level of the Medium 84 13* Inability of Vanadium to Substitute for Molybdenum In the Growth of Azotobacter vinelandii 85 v vi Indirect Demonstration of a Molybdenum Re­ quirement fqr Azotobacter vinelandii Grown oti NH^ “ 86 Comparison of the Intracellular Distribution and Uptake of and Mo" in Azotobacter vinelandii 87 Dialysis of the Tungsten and Molybdenum of the Azotobacter vinelandii 25,000 Times Gravity Supernatant Praction 88 M o " Dialysis Loss from the 25,000 Times Gravity Supernatant Fraction of Azotobacter vinelandii as a Result of Various Treatments 89 yyl85 j5ialysis Loss from the 25,000 Times Gravity Supernatant Fraction of Azotobacter vinelandii as a Result of Various^ Treatments 90 Distribution of Fe59, W185, and M o " by Direct Lysozyme Lysis of Azotobacter vinelandii 91 Chromium as a Non-competitive Inhibitor of Molybdenum in Azotobacter vinelandii 92 Chromium as a Non-competitive Inhibitor of Iron in Azotobacter vinelandii 93 Comparison of the Molybdenum Requirements of Azotobacter vinelandii strain 0 and Azotobacter chroococcum C44 94 Competitive Inhibition of Molybdenum by Tungsten in the Growth of Azotobacter chroococcum 95 Relative Uptake of M o " by Azotobacter chroococcum as a Function of Various Treatments and as Compared to Azotobacter vinelandii 96 99 Distribution of Mo by Direct Lysozyme Lysis of Azotobacter chroococcum 97 Effect of Various Ge/Si Ratios on M o " Up­ take in Azotobacter vinelandii 98 vii 31 27. Uptake of Si as a Function of Germanium and Molybdenum Levels in Azotobacter vinelandii 99 28. Uptake of Si31 by Azotobacter vinelandii as a Function of Germanium, Molybdenum, and Phosphorus Levels 100 31 29. Distribution of Si in Cell-Free Fractions of Azotobacter vinelandii 101 31 30. Distribution of Si by Direct Lysozyme Lysis of Azotobacter vinelandii 102 31. Germanium Inhibition of Azotobacter vine­ landii as Influenced by"Pbosphate Level 103 32. Half-Life of Cyclotron Produced Nitrogen-*-5 104 33. N -*-3 Incorporation by Various Systems 105 MOLYBDATE METABOLISM OF AZOTOBACTER INTRODUCTION Molybdenum has received attention in recent years as a metal component in various enzyme systems* It has been implicated with some certainty in xanthine oxidase, aldehyde oxidase, nitrate reductase, hydrogenase, and as an absolute requirement for various organisms fixing elemental nitrogen. Whether or not it is an actual in vivo component of these systems has not been established unequivocally. The strength of the evidence varies with the enzyme in question. It appears rather certain that nitrate reductase and xanthine oxidase are molybdoproteins, while the evidence for the others is less certain than could be desired. A few organisms are known which have the ability of fixing elemental nitrogen, that is, of using N2 gas as the sole nitrogen source. Azotobacter, an aerobic hetero- troph, is one of these organisms. It is known further that Azotobacter has a hydrogenase, can also reduce nitrate, and perhaps contains xanthine oxidase and aldehyde oxidase as well. It seemed, therefore, that Azotobacter might be an excellent organism in which to study the metabolism of molybdenum. 2 The study of the actual in vivo metabolism of a suspected enzymatic cofactor might be expected to reveal information which could not be obtained by the direct study of isolated enzyme systems. With information obtained by both approaches, one might be more likely to establish the means by which molybdenum facilitates various functions in the living cell. The requirement of molybdenum for nitrogen fixation offers an especially interesting challenge because the entire pathway of fixation has not yet been established, and thus the exact step(s) in which molybdenum is required and its exact function(s) are not known. Labeled molybdenum, under the right conditions, might possibly be utilized to localize the nitrogen fixing system as well as the other molybdenum containing enzymes. In this way one might identify at least a part of the nitrogenase system. Therefore the objective of this study was to examine the metabolism of molybdate, the form in which the molybdenum is supplied to the Azotobacter cell, and to de­ termine the nature of its disposition in the cell. It was hoped to be able to develop methods for determining the number and nature of the functional sites of molybdate utili­ zation. If this were possible, and If more than one site existed, and if one could assign a role to each of these sites by appropriate techniques, then one might be better able to ascribe unequivocal importance to molybdenum in individual in vivo enzyme systems. Furthermore, one might subsequently be able to purify toward the systems using QQ the molybdenum1757 label. This would be especially useful in the case of nitrogenase, an enzyme system which has thus far escaped purification. REVIEW OF THE LITERATURE Molybdenum, Tungsten, and Vanadium In Biological Systems Interest in the role of molybdenum has Increased greatly since the observation In 1950 by Bortels (8 ) that nitrogen fixation was enhanced by this element. A number of groups throughout the world are now actively engaged in Investigations of various systems in which molybdenum is a component. The early work from the time of Bortels centered around the role of molybdenum In nitrogen fixation. For example, In 1932 Birch-HIrshfeld (7) reported no response by Azotobacter to molybdenum when nitrate was the nitrogen source rather than elemental nitrogen. However, Burk and Horner (13) in 1939 were able to show a requirement for molybdenum when the cells were utilizing nitrate. The con­ centration of molybdenum required was less for cells utilizing nitrate than for nitrogen-gas-grown cells accord­ ing to Bortels (10). Thus, a requirement for molybdenum in nitrate reduction as well as in nitrogen fixation also became evident since, of all nitrogen sources tried by all Investigators, only the utilization of Ug and NO3 was molyb­ date dependent. The molybdenum requirement under various nitrogen sources is still undergoing active Investigation 4 (11,16,30,32,49). An interesting variation of the molybdate requirement by different species of Azotobacter was reported by Horner et al (26). A strain of A. vinelandii tested required much lower molybdate concentrations for optimum growth than various Azotobacter chroococcum strains. More recent work has been in the area of molybdenum as a cofactor in various partially purified enzyme systems. A recent review by Evans (17) describes the importance of molybdenum in nutrition as well as the role of the element in various enzymatic reactions. Mahler and Green (41) in an earlier review discussed the metallo-flavoproteins among which are found molybdenum containing systems. More specifically, Nason and Evans (56), and Nicholas and Nason (52,53) were responsible for the isolation of a molybdenum containing nitrate reductase capable of catalyzing the oxi­ dation of reduced TPN by nitrate. Intestinal xanthine oxidase was shown by Westerfeld and Richert (76) to be rather low in rats maintained on molybdenum free diets. Confirmation experiments and proof that molybdenum was an actual constituent of xanthine oxidase followed (21,57,61,70). Mahler, Mackler and Green (38,42) described an aldehyde oxidase enzyme dependent on molybdenum. Shug £t jal (65) also described a molybdo-enzyme, this time a hydrogenase from Clostridium pasteurianum.