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The Chemistry and Biochemistry of Nitrogen Fixation The Chemistry and Biochemistry of Nitrogen Fixation Edited by J. R. Postgate Universi~ of Sussex Falmer, Brighton Sussex PLENUM PRESS • London - New York ISBN-13: 978-1-4684-1820-0 e-ISBN-13: 978-1-4684-1818-7 001: 10.1007/978-1-4684-1818-7 Copyright © 1971 Plenum Publishing Company Ltd. Softcover reprint of the hardcover 15t edition 1971 Plenum Publishing Company Ltd. Davis House, 8 Scrubs Lane, Harlesden, London NWI0 6SE. U.S. Edition published by Plenum Publishing Corporation, 227 West 17th Street, New York, New York 10011. All Rights Reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher. SBN: 306-30459-7 Library of Congress Catalog Card Number: 70-161303 First Printing-December 1971 Second Printing-April 1976 CONTRIBUTORS R. H. Burris Department of Biochemistry, University of Wis consin, Madison 6, Wisconsin 53706, U.S.A. ]. Chatt A.R.C. Unit of Nitrogen Fz'xation, University of Sussex, Falmer, Brighton BNl 9Qj, Sussex. H.]. Evans Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, U.S.A. G.]. Leigh A.R.C. Unit of Nitrogen Fixation, University of Sussex, Falmer, Brighton BNl 9Qj, Sussex. ]. R. Postgate A.R.C. Unit of Nitrogen Fixation, Universzty of Sussex, Falmer, Brighton BNl 9QJ, Sussex. R. L. Richards A.R.C. Unit of Nitrogen Fixation, University of Sussex, Falmer, Brighton BNl 9QJ, Sussex. S. A. Russell Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, U.S.A. W. S. Silver Department of Life Sciences, Indiana State University, Terre Haute, Indiana 47809, U.S.A. P. W. Wilson Department of Bacteriology, University of Wis consin, Madison 6, Wisconsin 53706, U.S.A. M. G. Yates A.R.C. Unit of Nitrogen Fixation, University of Sussex, Falmer, Brighton BNl 9QJ, Sussex. v CONTENTS Preface Xl Chapter 1 The Background. Perry W. Wilson 1.1 Introduction 1 1.2 Properties of the Enzyme System in Azoto bacter 4 1.3 The Properties of the Enzyme System in Leguminous Plants 7 1.4 The Chemical Pathway 11 Chapter 2 Abiological Nitrogen Fixation. C. ]. Leigh 2.1 Introduction 19 2.2 The Properties of Dinitrogen 20 2.3 Modes of Reaction of Dinitrogen 26 2.4 A Possible Mechanism for Biological N 2 - Fixation 50 Chapter 3 Dinitrogen Complexes and Nitrogen Fixation. J. Chatt and R. L. Richards 3.1 Introduction 57 3.2 Terminal-Dinitrogen Complexes 65 3.3 Bridging-Dinitrogen Complexes 91 Chapter 4 Fixation by Free-Living Micro-Organisms: Enzymology. R. H. Burris 4.1 Introduction 106 4.2 Extraction, Purification and Properties of the Enzymes 1 09 4.3 Cofactors in N 2 Fixation 122 4.4 Reactions Catalyzed by Nitrogenase 128 4.5 Inhibitors of N2 Fixation 145 4.6 Kinetics of N2 Fixation 150 4.7 Metabolic Schemes for N2 Fixation 151 VII viii CONTENTS Chapter 5 Fixation By Free-Living Microbes: Physiology. J. R. Postgate 5.1 Introduction 161 5.2 The Reductive Character of N 2 -Fixation 163 5.3 The Need for ATP 177 5.4 The Need for Iron and Molybdenum 181 5.5 Conclusion: Some Unresolved Problems 185 Chapter 6 Physiological Chemistry of Symbiotic Nitrogen Fixation by Legumes. H. J. Evans and S. A. Russell 6.1 Introduction 191 6.2 The Symbiotic Association 193 6.3 Specific Mineral Requirements 196 6.4 Leghemoglobin 207 6.5 The Nitrogenase System 213 6.6 Electron Transport 229 6.7 Conclusions 236 Addendum 243 Chapter 7 Physiological Chemistry of Non-Leguminous Symbiosis. W. S. Silver 7.1 Introduction 246 7.2 Biological Aspects of Non-Leguminous Symbiosis 246 7.3 Physiological and Biochemical Aspects of Nitrogen Fixation In True Root-Nodule Symbiosis 254 7.4 Physiological and Biochemical Aspects of Nitrogen Fixation In Superficial Root Nodule Symbiosis 266 7.5 Physiological and Biochemical Aspects of Foliar Nitrogen Fixation 268 7.6 Physiological Ecology of N2 Fixation by Non-Legumes 270 7.7 Speculations on the Evolution of Symbiotic Nitrogen Fixation In Root-Nodule and Foliar Symbiosis of Non-Legumes 274 CONTENTS IX Appendix 1 A Review of Research by Soviet Scientists on the Biochemistry of Nitrogen Fixation. M. G. Yates Al.1 Introduction 283 Al.2 The Nitrogenase Reaction 286 A1.3 Enzymes Associated with Nitrogen Fixa- tion: Electron Transport 294 A1.4 Mechanisms of the Nitrogenase Reaction and the Nature of the Active Site 300 A1.5 Separation of Nitrogenase into its Com- ponent Fractions 303 A1.6 Control of Nitrogen Fixation in Vivo 304 A1.7 Conclusion 304 Appendix 2 The Acetylene Test for Nitrogenase.]. R. Postgate A2.1 Introduction 311 A2.2 Equipment 312 A2.3 Gases 312 A2.4 Tests of Soil, Water. and Tissue Samples 312 A2.5 Tests of Aerobic Micro-Organisms 313 A2.6 Tests of Facultative and Obligate Anaerobes 313 A2.7 Tests of Enzyme Preparations 314 A2.8 Interpretation 314 Subject Index 317 PREFACE Understanding of biological nitrogen fixation has advanced with impressive rapidity during the last decade. As befits a developing area of Science, these advances have uncovered information and raised questions which will have, and indeed have had, repercussions in numerous other branches of science and its applications. This 'information explosion', to use one of to-day's cant idioms, was initiated by the discovery, by a group of scientists working in the Central Research laboratories of Dupont de Nemours, U.S.A., of a reproducibly active, cell-free enzyme preparation from a nitrogen fixing bacterium. Full credit is due to them. But subsequent developments, albeit sometimes quite as impressive, have too often been marked by that familiar disorder of a developing field of research-the scramble to publish. It is a scramble which, at its best, may represent a laudable desire to inform colleagues of the latest developments; yet which too easily develops into an undignified rush for priority, wherewith to impress one's Board of Directors or Grant-giving Institution. This, in miniature, is the tragedy of scientific research to-day: desire for credit causes research to be published in little bulletins, notes and preliminary communications, so that only those intimately involved in the field really know what is happening (and even they may well not see the forest for the trees). Those outside the field, or working in peripheral areas, may glean something of what is going on from reviews and fragments presented at meetings, but the broad pattern of development is often elusive. This book is an attempt to correct the situation within its own particular field. An explanation (some might even sayan apology) is to-day necessary from everyone who presumes to add to the mounting deluge of scientific publications; for this book I offer the view that it is intended for the informed outsider. I asked con tributors to stand back a little from the subject and to describe what the real advances of the last decade had been. What were they? What was their impact on our understanding of this particular subject? How did they influence our background knowledge of chemistry, Xl xu PREFACE biochemistry or physiology? The book was intended to be com plementary to existing works on nitrogen fixation: an advanced but readily comprehensible survey of the last decade's innovations for students, teachers and research workers who would be reasonably well-informed about its background_ Contributors, all recognized authorities in their particular fields, were asked specifically not to write reviews: there was no obligation to cite all known references bearing on a given aspect of the subject; historical continuity could be ignored and even the desire to be in all ways up-to-date could be resisted. Painstaking and exhaustive reviews of nitrogen fixation, in its biochemical, chemical and biological aspects, exist in plenty; contributors were asked to present their material as a survey which, while in no way 'talking down', would be useful and comprehensible to scientists whose training and interests would be broader and might range from purely chemical to wholly biological. I thank my fellow contributors for interpreting my intentions so effectively. I am aware that some have not been able to resist adding their latest 'stop-press' item; that others, having been aware of an unseemly squabble for priority, may have felt it necessary to describe the history of a certain advance in a degree detail which may seem strange to those not involved; yet others have been overtaken by events (my own contribution contains information about heterocysts and nitrogen fixation by blue-green algae which became obsolete in press; fortunately Professor Burris's chapter came in later and * amends it). I have left these human touches intentionally, in the hope that the synoptic view taken by the book as a whole will compensate for the occasional idiosyncracy. (And what could be more tedious than contributions devoid of idiosyncracy?) A final word about the first contributor. In 1940 Perry Wilson published 'The Biochemistry of Symbiotic Nitrogen Fixation', a seminal work which served as a springboard for a great deal of meaningful work in this field. The decade which the present volume covers ended with the thirtieth anniversary of Wilson's book and, at an early stage, we contributors had intended to dedicate our book to Perry Wilson as a kind of festschrift. Illness led to a re-shuffle of contributors and I found myself calling upon Perry, not to stand and be admired, but to do some more work. So our book is graced by Perry's opening chapter. Our intention to honour him may have been thwarted but our esteem for his monumental contribution to the subject remains unaltered. We * Compare pages 1 77 and 149 PREFACE Xlll wished to do him formal honour; in the event we could not. But it is a great pleasure, and in some ways it feels more natural, to have him here with us. John Postgate University of Sussex January, 1971 Acknowledgement. Four contributors (G. J. Leigh, J. Chatt, J. R. Postgate, M. G. Yates) are employees of the British Agricultural Research Council at its Unit of Nitrogen Fixation, University of Sussex. They acknowledge permission to contribute and emphasize that all opinions and interpretations expressed are their own and in no way commit the Council.