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THE MICROBIAL FERROUSWHEEL: IRON CYCLING INTERRESTRIAL, FRESHWATER,

AND MARINE ENVIRONMENTS

Hosted byDavid Emerson, Eric Roden andBenjamin Twining

MICROBIOLOGY
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Frontiers in Microbiology December 2012 | The microbial ferrous wheel: iron cycling in terrestrial, freshwater, and marine environments | 1

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ISSN1664-8714

ISBN978-2-88919-074-4

DOI 10.3389/978-2-88919-074-4
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Hosted By:David Emerson, Bigelow Laboratory for Ocean Sciences, USAEric Roden, University of Wisconsin-Madison, USABenjamin Twining, Bigelow Laboratory for Ocean Sciences, USA

In the past 15 years, there has been steady growth inwork relating to the microbial iron cycle. It is now well

established that in anaerobic environments couplingof organic matter utilization to Fe reduction is amajor pathway for anaerobic respiration. In iron-richcircumneutral environments that exist at oxic-anoxicboundaries, significant progress has been made indemonstrating that unique groups of microbes cangrow either aerobically or anaerobically using Fe as aprimary energy source. Likewise, in high iron acidicenvironments, progress has been made in the studyof communities of microbes that oxidize iron, andin understanding the details of how certain of theseorganisms gain energy from Fe-oxidation. On the ironscarcity side, it is now appreciated that in large areasof the open ocean Fe is a key limiting nutrient; thus, agreat deal of research is going into understanding thestrategies microbial cells, principally phytoplankton, useto acquire iron, and how the iron cycle may impact othernutrient cycles. Finally, due to its abundance, iron has

played an important role in the evolution of Earths primary biogeochemical cycles throughtime.

THE MICROBIAL FERROUS WHEEL:

IRON CYCLING IN TERRESTRIAL,

FRESHWATER, AND MARINE

ENVIRONMENTS

Figure reproduced from Kozubal et al.

(2012) (see p. 161).
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The aim of this Research Topic is to gather contributions from scientists working indiverse disciplines who have common interests in iron cycling at the process level, and atthe organismal level, both from the perspective of Fe as an energy source, or as a limitingnutrient for primary productivity in the ocean. The range of disciplines may include:geomicrobiologists, microbial ecologists, microbial physiologists, biological oceanographers,and biogeochemists. Articles can be original research, techniques, reviews, or synthesis papers.

An overarching goal is to demonstrate the environmental breadth of the iron cycle, and fosterunderstanding between different scientific communities who may not always be aware of oneanothers work.

Suggested Topics:- Microbial Fe oxidation at neutral and acidic pH- Microbial Fe reduction- Ecosystem coupling of Fe-oxidation and reduction- Iron availability in nutrient limited environments (or iron as a limiting nutrient in

marine ecosystems)- Cellular iron acquisition strategies of environmental importance- Advanced techniques for studying iron biogeochemistry- Paleo-microbiology of Fe-cycling
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Table of Contents

06 The Microbial Ferrous Wheel: Iron Cycling in Terrestrial, Freshwater, and

Marine Environments

David Emerson, Eric Roden and Benjamin S. Twining

08 The Influence of Extracellular Superoxide on Iron Redox Chemistry and

Bioavailability to Aquatic Microorganisms

Andrew L. Rose

29 The Organic Complexation of iron in the Marine Environment: A Review

Martha Gledhill and Kristen N Buck46 Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella

Oneidensis MR-1

Liang Shi, Kevin M. Rosso, Tomas A. Clarke, David J. Richardson, John M. Zachara

and James K. Fredrickson

56 Abiotic and Microbial Interactions During Anaerobic Transformations of Fe(II)

and NOX

Flynn Picardal

63 Redox Transformations of Iron at Extremely Low pH: Fundamental and

Applied Aspects

D. Barrie Johnson, Tadayoshi Kanao and Sabrina Hedrich

76 Toward a Mechanistic Understanding of Anaerobic Nitrate-Dependent IronOxidation: Balancing Electron Uptake and Detoxification

Hans K. Carlson, Iain C. Clark, Ryan A. Melnyk and John D. Coates

82 The Importance of Kinetics and Redox in the Biogeochemical Cycling of Iron

in the Surface Ocean

Peter L. Croot and Maija I. Heller

97 Reconstruction of Extracellular Respiratory Pathways for Iron(III) Reduction in

Shewanella Oneidensis Strain MR-1

Dan Coursolle and Jeffrey A. Gralnick

108 Iron-Based Microbial Ecosystem on and Below the Seafloor: A Case Study

of Hydrothermal Fields of the Southern Mariana Trough

Shingo Kato, Kentaro Nakamura, Tomohiro Toki, Jun-ichiro Ishibashi, Urumu Tsunogai,

Akinori Hirota, Moriya Ohkuma and Akihiko Yamagishi

122 Isolation of PhyllosilicateIron Redox Cycling Microorganisms from an

IlliteSmectite Rich Hydromorphic Soil

Evgenya Shelobolina, Hiromi Konishi, Huifang Xu, Jason Benzine, Mai Yia Xiong,

Tao Wu, Marco Blthe and Eric Roden

132 Microbial Iron(II) Oxidation in Littoral Freshwater Lake Sediment: The Potential

for Competition between Phototrophic vs. Nitrate-Reducing Iron(II)-Oxidizers

E. D. Melton, C. Schmidt and A. Kappler
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144 In situ Spectroscopy on Intact Leptospirillum Ferrooxidans Reveals that

Reduced Cytochrome 579 is an Obligatory Intermediate in the Aerobic Iron

Respiratory Chain

Robert C. Blake and Megan N. Griff

154 Microbial Iron Cycling in Acidic Geothermal Springs of Yellowstone National

Park: Integrating Molecular Surveys, Geochemical Processes, and Isolation of

Novel Fe-Active MicroorganismsMark A. Kozubal, Richard E. Macur, Zackary J. Jay, Jacob P. Beam, Stephanie A.

Malfatti, Susannah G. Tringe, Benjamin D. Kocar, Thomas Borch and William P. Inskeep

170 Identification and Characterization of MtoA: A Decaheme c-Type Cytochrome

of the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1

Juan Liu, Zheming Wang, Sara M. Belchik, Marcus J. Edwards, Chongxuan Liu,

David W. Kennedy, Eric D. Merkley, Mary S. Lipton, Julea N. Butt, David J.

Richardson, John M. Zachara, James K. Fredrickson, Kevin M. Rosso and Liang Shi

181 Mineralogy of Iron Microbial Mats from Loihi Seamount

Brandy M. Toner, Thelma S. Berqu, F. Marc Michel, Jeffry V. Sorensen,

Alexis S. Templeton and Katrina J. Edwards

199 The Microbial Ferrous Wheel in a Neutral pH Groundwater SeepEric E. Roden, Joyce M. McBeth, Marco Blthe, Elizabeth M. Percak-Dennett,

Emily J. Fleming, Rebecca R. Holyoke, George W. Luther, David Emerson

and Juergen Schieber
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EDITORIALpublished: 31 October 2012

doi: 10.3389/fmicb.2012.00383

The microbial ferrous wheel: iron cycling in terrestrial,freshwater, and marine environments

David Emerson1*,Eric Roden2 andBenjamin S. Twining1

1 Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA2 Department of Geoscience, University of Wisconsin, Madison, WI, USA

*Correspondence: [email protected]

Edited by:

Bradley M. Tebo, Oregon Health and Science University, USA

Reviewed by:

Bradley M. Tebo, Oregon Health and Science University, USA

Were Oscar Wilde a devotee of iron biogeochemistry in thetwenty-first century (hard as that might be to imagine), he might

remark that iron is the most ironic of elements. Those inter-

ested in microbes that carry out life-sustaining iron-coupled

redox reactions like to point out that iron is, after oxy-

gen, the most abundant redox-active element in the Earthscrust. However, those studying oceanic phytoplankton regard

iron as a nutrient that occurs at such vanishingly low con-centrations in the surface ocean that it limits the growth of

algae in more than 40% of the global ocean. This is becauseat the pH of seawater oxygen promotes the rapid oxida-

tion of soluble ferrous iron to insoluble ferric iron oxyhy-

droxides that precipitate and sink out of the water column.

As a result, while many marine microbes, and especially thephotosynthetic ones, have developed finely-tuned mechanisms

for acquiring iron, total primary productivity can be limitedby iron.

At oceanic hydrothermal vents, and in terrestrial habitats,

iron is not a limiting nutrient. At many oxic-anoxic inter-facial habitats, not only is iron not limiting, but it is so

abundant that lithotrophic microbes can use it as an elec-

tron source to sustain growth, and form robust communitiesof iron-oxidizing chemolithoautotrophs. In more acidic con-

ditions, such as certain hot springs and acid mine drainage

systems, ferrous iron is more stable, concentrations can be

in the millmolar range, and specific communities of archaea

and bacteria that use iron as an energy source can flour-ish. Iron-oxidizing microbes are not limited to aerobic habi-

tats, but can also oxidize iron under anaerobic conditions by

coupling the oxidation to either anoxygenic photosynthesis or

nitrate reduction. Nor does it appear that they are limited to

only utilizing soluble ferrous iron as an energy source, butcan also acquire iron from insoluble minerals that containreduced iron.

But iron-oxidation is only one-half of the equation. The uti-

lization of ferric iron, principally in the form of Fe-oxides, to

carry out anaerobic respiration is well established as an importantpathway for organic carbon metabolism in anaerobic habitats.

Furthermore, model organisms such asShewanellaandGeobacterare utilized to study the biochemical mechanisms of Fe-reduction,

and from this we have learned a good deal about processes

involved in extracellular electron transfer. Taken as a whole, it

is apparent that the iron cycle is a remarkably complex process,

dependent upon a wide range of chemical interactions, habitat

types and groups of microbes that link it to all of Earths otherimportant biogeochemical cycles.

In this special topic issue we have gathered contributions

from scientists working in diverse disciplines who have commoninterests in iron cycling at the process level and at the organ-

ismal level, from the perspective of iron as an energy sourceor as a limiting nutrient for primary productivity in the ocean.

The hope is that bringing together seemingly disparate lines of

research under one cover will result in a more global under-

standing of the iron cycle, and perhaps draw new insight intothe connections within the cycle. We were very fortunate to

enlist a varied and talented group of authors to contribute awide range of articles. In total, 16 papers have been included,

with a mixture of 9 original research articles, 6 reviews, and 1

perspective.

Aspects of iron cycling in the open ocean are covered by

reviews on organic complexation (Gledhill and Buck,2012) andon the role of superoxide dismutase (Rose, 2012), as well asin a research article on the role of weak iron-binding ligands

in the ocean by Croot and Heller (2012). Oxygen-dependent

iron oxidation at circumneutral pH is addressed in a research

paper on a potential mechanism for iron oxidation by Liuet al. (2012), a research paper on mineralogy of biogenically-

formed oxides at a hydrothermal vent (Toner et al.,2012), anda review of iron-based ecosystems associated with hydrother-mal vents and the subsurface in the Pacific by Kato et al.

(2012). Iron-cycling in acidic systems is reviewed byJohnsonet al.(2012), and original research on a unique iron-rich acidic

ecosystem in Yellowstone National Park is presented byKozubal

et al. (2012). A novel spectroscopic technique for biochemi-cal analysis of iron oxidation in Leptospirillum ferroxidans is

contributed byBlake and Griff(2012). Microbial utilization of

iron under anaerobic conditions is dealt with in a review ofmechanisms for iron reduction by Shi et al. (2012). Picardal

(2012) reviews abiotic and microbial interactions of anaero-bic iron oxidation and Carlson et al. (2012) provide an inter-

esting perspective piece on nitrate-dependent iron oxidation.

Original research on iron reduction in Shewanella is presented

by Coursolle and Gralnick (2012), and competition amongphototrophic and nitrate-dependent iron-oxidizing microbes is

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Emerson et al. The microbial ferrous wheel

addressed by Melton et al. (2012). Finally, original work fromthe laboratory of Eric Roden investigates redox cycling in a typ-

ical freshwater iron-rich stream (Roden et al., 2012), as well asthe capacity for microbes to use iron minerals as an iron source

(Shelobolina et al.,2012).

Taken together these papers present an overview of researchon iron from a range of perspectives that indicates the breadth

of work that has been done and provides insight into the manyexciting avenues of research that continue to enhance our under-

standing of the iron cycle in nature.

REFERENCESBlake, I. I. R. C., and Griff, M.

N. (2012). In situ spectroscopy

on intact Leptospirillum ferrooxidans

reveals that reduced cytochrome

579 is an obligatory intermedi-

ate in the aerobic iron respiratory

chain. Front. Microbio. 3:136. doi:

10.3389/fmicb.2012.00136

Carlson, H. K., Clark, I. C., Melnyk,

R. A., and Coates, J. D. (2012).

Toward a mechanistic understand-

ing of anaerobic nitrate-dependent

iron oxidation: balancing elec-

tron uptake and detoxification.

Front. Microbio. 3 :57. doi:

10.3389/fmicb.2012.00057Coursolle, D., and Gralnick, J.

A. (2012). Reconstruction of

extracellular respiratory path-

ways for iron(III). Reduction

in Shewanella oneidensis strain

MR-1. Front. Microbio. 3:56. doi:

10.3389/fmicb.2012.00056

Croot, P. L., and Heller, M. I. (2012).

The importance of kinetics and

redox in the biogeochemical

cycling of iron in the surface

ocean. Front. Microbio. 3:219. doi:

10.3389/fmicb.2012.00219

Gledhill, M., and Buck, K. N. (2012).

The organic complexation of iron

in the marine environment: areview. Front. Microbio. 3:69. doi:

10.3389/fmicb.2012.00069

Johnson, D. B., Kanao, T., and Hedrich,

S. (2012). Redox transforma-

tions of iron at extremely low

pH: fundamental and applied

aspects. Front. Microbio. 3:96. doi:

10.3389/fmicb.2012.00096

Kato, S., Nakamura, K., Toki, T.,

Ishibashi, J.-I., Tsunogai, U., Hirota,

A., et al. (2012). Iron-based micro-

bial ecosystem on and below the

seafloor: a case study of hydrother-

mal fields of the southern mariana

trough. Front. Microbio. 3:89. doi:

10.3389/fmicb.2012.00089

Kozubal, M. A., Macur, R. E., Jay,

Z. J., Beam, J. P., Malfatti, S. A.,

Tringe, S. G., et al. (2012). Microbialiron cycling in acidic geothermal

springs of Yellowstone National

Park: integrating molecular surveys,

geochemical processes, and isola-

tion of novel Fe-active microorgan-

isms. Front. Microbio. 3:109. doi:

10.3389/fmicb.2012.00109

Liu, J., Wang, Z., Belchik, S. M.,

Edwards, M. J., Liu, C., Kennedy, D.

W., et al. (2012). Identification and

characterization of MtoA: a dec-

aheme c-type cytochrome of the

neutrophilic Fe(II)-oxidizing bac-

terium Si deroxydans lithotrophicus

ES-1. Front. Microbio. 3:37. doi:

10.3389/fmicb.2012.00037Melton, E. D., Schmidt, C., and

Kappler, A. (2012). Microbial

iron(II). Oxidation in lit-

toral freshwater lake sediment:

the potential for competition

between phototrophic vs. nitrate-

reducing iron(II)-oxidizers.

Front. Microbio. 3:197. doi:

10.3389/fmicb.2012.00197

Picardal, F. (2012). Abiotic and micro-

bial interactions during anaero-

bic transformations of Fe(II) and

NOx. Front. Microbio. 3:112. doi:

10.3389/fmicb.2012.00112

Roden, E. E., McBeth, J. M., Blthe, M.,

Percak-Dennett, E. M., Fleming,

E. J., Holyoke, R. R., et al. (2012).

The microbial ferrous wheel

in a neutral pH groundwaterseep. Front. Microbio. 3:172. doi:

10.3389/fmicb.2012.00172

Rose, A. L. (2012). The influence of

extracellular superoxide on iron

redox chemistry and bioavailabil-

ity to aquatic microorganisms.


10.3389/fmicb.2012.00124

Shelobolina, E., Konishi, H., Xu,

H., Benzine, J., Xiong, M. Y.,

Wu, T., et al. (2012). Isolation of

phyllosilicateiron redox cycling

microorganisms from an illite

smectite rich hydromorphic

soil. Front. Microbio. 3:134. doi:

10.3389/fmicb.2012.00134Shi, L., Rosso, K. M., Clarke, T. A.,

Richardson, D. J., Zachara, J. M.,

and Fredrickson, J. K. (2012).

Molecular underpinnings of Fe(III)

oxide reduction by Shewanella

oneidensis MR-1. Front. Microbio.

3:50. doi: 10.3389/fmicb.2012.

00050

Toner, B. M., Berqu, T. S., Michel,

F. M., S orensen, J. V., Templeton,

A. S., and Edwards, K. J. (2012).

Mineralogy of iron microbial

mats from loihi seamount.


10.3389/fmicb.2012.00118

Received: 02 October 2012; accepted:

14 October 2012; published online: 31

October 2012.Citation: Emerson D, Roden E and

Twining BS (2012) The microbial fer-

rous wheel: iron cycling in terrestrial,

freshwater, and marine environments.

Front. Microbio. 3:383. doi: 10.3389/

fmicb.2012.00383

This article was submitted to Frontiers in

Microbiological Chemistr y, a specialty of

Frontiers in Microbiology.

Copyright 2012 Emerson, Roden and

Twining. This is an open-access arti-

cle distributed under the terms of the

Creative Commons Attribution License,

which permits use, distribution and

reproduction in other forums, provided

the original authors and source are cred-ited and subject to any copyright notices

concerning any third-party graphics etc.

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REVIEWARTICLEpublished: 11 April 2012

doi: 10.3389/fmicb.2012.00124

The influence of extracellular superoxide on iron redoxchemistry and bioavailability to aquatic microorganisms

Andrew L. Rose*

Southern Cross GeoScience, Southern Cross University, Lismore, NSW, Australia

Edited by:

BenjaminTwining, Bigelow

Laboratory for Ocean Sciences, USA

Reviewed by:

Colleen Hansel, Harvard University,

USA

Kai Waldemar Finster, Aarhus

University, Denmark

Yeala Shaked, Hebrew University,

Israel

*Correspondence:

Andrew L. Rose, Southern Cross

GeoScience, Southern Cross

University, PO Box 157, Lismore,

NSW 2480, Australia.

e-mail: [email protected]

Superoxide, the one-electron reduced form of dioxygen, is produced in the extracellular

milieu of aquatic microbes through a range of abiotic chemical processes and also by

microbes themselves. Due to its ability to promote both oxidative and reductive reac-

tions, superoxide may have a profound impact on the redox state of iron, potentially

influencing iron solubility, complex speciation, and bioavailability. The interplay between

iron, superoxide, and oxygen may also produce a cascade of other highly reactive tran-

sients in oxygenated natural waters. For microbes, the overall effect of reactions between

superoxide and iron may be deleterious or beneficial, depending on the organism and

its chemical environment. Here I critically discuss recent advances in understanding: (i)

sources of extracellular superoxide in natural waters, with a particular emphasis on micro-

bial generation; (ii) the chemistry of reactions between superoxide and iron; and (iii) the

influence of these processes on iron bioavailability and microbial iron nutrition.Keywords: iron, superoxide, bioavailability

INTRODUCTION

Superoxide (O2), an anion resulting from the transfer of one-electron to a molecule of oxygen, was first identified by EdwardNeuman, a postdoctoral researcher working with Linus Pauling,in 1934 (Neuman,1934) as part of Paulings ongoing interest inthe nature of the chemical bond(Pauling, 1979). In aqueous solu-tions, superoxide exists in equilibrium with its conjugate acid,the hydroperoxyl (or perhydroxyl) radical (HOO)(Bielski et al.,1985). The anion dominates the equilibrium in solutions whose

pH is above the pKa = 4.8 (Bielski et al.,1985). This is the casein the majority of natural surface waters, and at pH 8.1, whichis typical of marine waters and many carbonate-buffered freshwa-ters, superoxide is 2000-fold more abundant than hydroperoxyl.To distinguish between the superoxide anion and total superox-ide (i.e., both O2 and HOO

), I will represent total superoxideas O2 .

O2 has two chemical properties that produce a special rela-tionship with Fe in natural aquatic environments. First, it may actas both a reductant (via its oxidation to O2) and oxidant (via itsreduction to H2O2), with the redox potentials of the O2/O

2 and

O2 /H2O2couples in natural waters around neutral pH poised insuchawaythatO2 is thermodynamically able to reactwith a vast

range of Fe complexes(Pierre et al.,2002). The second propertyarises from its unusual electronic structure. Despite possessing anodd number of valence shell electrons, the O2 anion is resonancestabilized by virtue of itssymmetry andexhibitsvery little free radi-calcharacter(Pauling, 1931; Neuman, 1934; Sawyer, 1991);henceIfollow the convention here of omitting the free radical symbol andrepresenting the superoxide anion as O2. Thus in natural watersaround pH 8 where the relatively unreactive O2 dominates overHOO, and in the absence of other suitably reactive partners, O2has a half-life of tens of seconds to hours and has been measured toaccumulate to typical concentrations in the range of 101000pM

(Rose et al., 2008b, 2010; Hansard et al., 2010; Shaked et al., 2010).These lifetimes are sufficiently long to ensure that O2 can diffusewell away from the site of its production, enabling it to influencelocal redox chemistry on a spatial scale that is biologically signif-icant, while typical concentrations are sufficiently high to ensurethat it can react at environmentally relevant rates. It is this criticalbalance between longevity and reactivity that renders O2 one ofa select few extracellular reducing agents in natural waters thatare able to exert a biologically significant influence on local redox

chemistry. The combination of the overlapping redox potentialsof the O2/O

2 couple with the redox potentials of wide range of

couples of Fe(II)/Fe(III) species, and the ability of O2 to persistat sufficiently high concentrations, ensures that the fates of O2and Fe are often closely tied in environmental systems, and also inmany intracellular biological systems, to the extent that they havebeen called partners in crime (Liochev and Fridovich,1999).

The existence of O2 has thus been known for nearly 90 years,initially being studied largely as a chemical curiosity due to itsunusual electronic structure. In the 1960s, its importance in theaqueous intracellular environment was established with the dis-coveryof superoxide dismutase (SOD),an enzyme whose role is todestroy O2 . The biological importance of O

2 has been further

reinforced by the discovery that SOD appears essential in aerobicorganisms(McCord et al., 1971), and is found in nearly all aerobicforms of life(Wolfe-Simon et al., 2005) as well as many anaerobes(Gregory et al., 1978). Production of O2 in the intracellular envi-ronment, its reactions with Fe, and effects on biological processes,have consequently been extensively studied over the past 50 years(see, for example,Sawyer and Gibian,1979;Sawyer and Valen-tine,1981;Fridovich,1986;Afanasev,1989;Winterbourn,1995).However it is only since the 1980s that the potential role of O2in the aqueous extracellular environment has been examined indetail.Baxter and Carey(1983)established that O2 was formed

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Rose Extracellular superoxide and iron chemistry

during photolysisof humic substances in naturalwaters, while fur-ther pioneering work by Zika (Petasne and Zika,1987;Micinskiet al.,1993) andZafiriou(1990)established that O2 occurs inmarine waters, where it may participate in reactions with a rangeof biologically important compounds. There has been renewedinterest in the role of O2 as a redox agent in natural waters inthe last decade primarily because the development of new highly

sensitive,rapid,and convenient analytical techniques (e.g., chemi-luminescence of MCLA and its derivatives, with detection limitson the order of a few picomolar; Nakano et al.,1986;Nakano,1998;Teranishi,2007), has allowed determination of O2 con-centrations (Rose et al.,2008a) and production rates (Godrantet al.,2009;Milne et al.,2009) at environmentally relevant values.The role of O2 was first accounted for in models of Fe specia-tion and bioavailability in natural waters byMiller et al.(1995),with increasing recognition of its potential importance reflectedby inclusion of reactions between O2 and Fe in several morerecent models(Weber et al.,2005,2007;Fan,2008).

The primary purpose of this paper is to provide a detailedoverview of the ways in which O2 in the external milieu of

microorganisms can modulate the chemical speciation of Fe, andtherebyinfluenceitsbiologicalavailability.Whiledrawingonsomeof the pioneering work on aqueous O2 chemistry in the intra-cellular environment (including its reactions with Fe), the scopeof the paper will be limited primarily to the extracellular environ-ment in natural waters. In addition, while the paper is intendedto consider the influence of extracellular superoxide on iron redoxchemistry and bioavailability to aquatic microorganisms in all nat-ural waters, it is relatively biased toward marine systems, becauseit is in these systems that most recent advances on the subject haveoccurred. While many other aspects of O2 chemistry in naturalwaters are still being unraveled, including its role in cycles of otherelements, this paper will consider only the role of O 2 in mod-

ulating Fe chemistry. In particular, the following issues will beaddressed with an attempt to identify major knowledge gaps andcritical research questions where possible:

1. How is O2 produced in the extracellular milieu in naturalwaters?

2. By what mechanisms, and to what degree, can O2 modulateFe chemistry in the extracellular environment?

3. How can O2 influence Fe bioavailability to microorganismsin aquatic environments, through direct and indirect means?

SOURCES OF EXTRACELLULAR SUPEROXIDE IN NATURAL

WATERS

OVERVIEWTheoretically, O2 can be produced via either the one-electronreduction of O2or the one-electron oxidation of H2O2. In prac-tice, reduction of O2appears to be the dominant pathway for O

2

production in natural waters, though oxidation of H2O2has beenshown to occur under some conditions (Moffett and Zafiriou,1990). Like O2, the dioxygen molecule also possesses an unusualelectronic structure, with its lowest energetic state (ground state)possessing two unpaired electrons with parallel spins, i.e., itsground state is a triplet state biradical (Sawyer, 1991). Quan-tum mechanical restrictions (the Pauli exclusion principle) dictate

that, under typical environmental conditions, triplet state dioxy-gen can react only extremely slowly with molecules possessingsinglet state electronic configurations (e.g., most organic com-pounds), but much more readily with free radicals (e.g., organicradicals and transition metal ions), such that the reduction ofdioxygen must proceed primarily via single electron transfer steps(Fridovich, 1998). This implies that reduction of dioxygen should

always result in O

2 production.Attempting to identify sources of O2 in oxygenated watersis complicated, because the redox cycling of a range of relativelylabile redox couples are intimately coupled in oxygenated nat-ural waters; thus, trying to establish which redox reaction governsthe system behavior is like the chicken and egg question ofwhich came first. A common property of these labile redox-activecompounds (LRACs) is that they are sufficiently labile to acceptand donate their cargo of electrons, but also sufficiently long-lived to enable transport on biologically relevant spatial scales.Three major processes result in the occurrence of reduced LRACsin oxygenated surface waters: abiotic photochemistry, biologi-cal activity, and transport of reduced LRACs into (at least par-

tially) oxygenated surface waters from other environments (e.g.,at sediment-water interfaces, discharge of anoxic groundwaters,rainwater deposition, etc.). In the case of biological processes, theultimate source of electrons is usually (but not always) water;in the case of abiotic photochemistry it is usually organic com-pounds, which are themselves often (although not always) derivedfrom biological activity. Biological reduction in oxygenated watersis also predominantly powered ultimately via solar energy (eitherthrough photosynthesis, or respiration of reduced substrates thathave been previously been produced by photosynthesis). In oxy-genated waters, O2 can then be produced by reduction of O2directly through one of these processes, or through reductionof another LRAC that then reacts with O2 to yield O

2 . Thus

the vast majority of O

2 production in natural waters is dri-ven ultimately by solar radiation, but the path by which thissolar energy ultimately induces reduction of O2 to O

2 varies

(Figure 1).The main pathways for reduction of O2 to O

2 can thus be

broadly grouped into abiotic thermal (dark) processes, abioticphotochemical processes, and biologically mediated processes.Until recently, abiotic photochemistry in surface waters wasthought to be the major pathway for O2 production in theextracellular environment(Cooper et al.,1989). A growing bodyof work has shown that aquatic microorganisms can also pro-duce O2 extracellularly, and that the flux via this pathway maybe at least as great as that via abiotic photochemistry (Hansard

et al., 2010; Rose et al., 2010). However abiotic photochem-istry is still likely to result in a non-negligible contribution tototal extracellular superoxide production (ESP) in most sunlitwaters, and in some cases may be the dominant source. Thecontribution of abiotic thermal production (through oxygena-tion of reduced LRACs) to overall rates of ESP in natural watersis unknown, although the observation that filtration to removecells (and other particulates) does not always completely inhibitESP (e.g.,Rose et al.,2010) suggests that it may be substantial insome environments. These pathways are now considered in moredetail.

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LRACox

LRACred

O2 O2

CELL

MEMB-RANE

oxygen-

reducing

enzyme

other

reductase

enzyme

NOM

O2

B

DE

C

F A

SUN-LIGHT

DIFFUSION

FIGURE 1 | Major sources of O

2 in natural waters. (A)Abiotic,

photochemical oxidation of natural organic matter (NOM) with concomitant

reduction of O2.(B)Biological reduction of O2in the extracellular milieu via

cell surface enzymes.(C)Biological reduction of oxidized labile redox-active

compounds (LRACox) in the extracellular milieu to form reduced labile

redox-active compounds (LRACred) that subsequently reduce O2 to O

2 .(D)

Biological release of LRACred into the extracellular milieu that subsequentlyreduce O2to O

2 .(E) Diffusion of LRACredproduced under suboxic

conditions into more oxygenated waters. (F) The O2/O

2 couple readily

exchanges electrons with a range of other labile redox-active compounds in

the extracellular milieu. Decay pathways for O

2 are not shown.

ABIOTIC THERMAL (DARK) PRODUCTION

O2 can theoretically be produced by oxygenation of a range ofreduced LRACs that may occur in natural waters. The oxygenationof reduced metals [e.g., Fe(II), Mn(II), V(II), Cr(II), Cu(I), andCo(II)] has been proposed to generally occur via the HaberWeissmechanism(Haber and Weiss,1934), resulting in the productionof O2 after the first step:

Mn+ + O2 M(n+1)+

+ O2 (1)

WhereMn+ represents the reduced formof the metal andM(n+1)+

its oxidized form.Howeveritisnotcertainthattheseelectrontransferstepsalways

resultin release of thefree intermediate,as O2 andperoxide anions

(O22 )both possess coordinative properties and may potentiallyremain bound in the inner coordination sphere of the oxidizedmetal center. Using computational chemistry methods, Rosso andMorgan (2002)concluded that V2+, Co2+, and Fe2+ were likely

to react with O2 via an outer-sphere mechanism, such that O2

would be formed outside the inner coordination sphere (and pre-sumably be able to diffuse into the bulk medium as the free anion),while Mn2+, Cr2+, and hydrolyzed Fe(II) species were suggestedto react with O2via an inner-sphere process, such that O

2 would

initially be coordinated to the metal center (Rosso and Morgan,2002). Fe(II) complexed by EDTA and similar ligands also appears

to react with O2via an inner-sphere process (Zang and van Eldik,1990;Seibig and van Eldik,1997).Whether O2 formed in this way could subsequently partici-

pate in other reactions likely depends on how rapidly it can escapefrom the inner coordination sphere after electron transfer is com-plete. It is unclear how Fe(II) in natural waters might behave, butcomplexation of Fe(II) by natural organic matter (NOM) appearsto substantially modify its oxygenation kinetics (e.g., Rose andWaite, 2003a and referencestherein), suggestingthat further inves-tigation of this issue is needed, ideally through both experimentalandcomputational means.Hereafter, it willbe assumed that reduc-tion of O2 ultimately results in production of unbound O

2 ,

even if it is initially formed in the inner coordination sphere of

a metal complex. However, this uncertainty in our understandingof O2 behavior needs to be resolved in order to fully evaluate therelationship between O2 and Fe bioavailabilty in natural waters.

Other reduced LRACs in natural waters include reduced sulfurspecies (RSS) such as thiols andsulfides,and some reduced organicmoieties. While the contribution of such species to ESP has not yetbeen evaluated, RSSmay be found at measurable concentrations inoxic waters (Bowles et al., 2003; Luther and Rickard, 2005; Rickardand Luther,2006) and at least some RSS yield O2 as a resultof oxygenation (Rao et al.,1990). Certain redox-active moietieswithin NOM also react readily with oxygen (Ratasuk and Nanny,2007; Aeschbacheretal., 2010),potentiallyyieldingO 2 (discussedfurther in the following section). The oxygenation chemistry (par-

ticularly thermodynamics) of a range of major LRACs present innatural waters has recently been described in considerable detailbyLuther(2010), while the thermodynamics of the Fe/O2/O

2

system is discussed in detail by Pierre et al. (Pierre and Fontecave,1999;Pierre et al.,2002).

PHOTOCHEMICAL PRODUCTION

Measured rates of photochemical O2 production in naturalmarine waters have typically been in the range of 0.3500nMh1 (Petasne and Zika, 1987;Micinski et al., 1993;Shakedet al.,2010). Rates of O2 production in natural freshwaters havenot been reported in the literature, although laboratory exper-iments using simulated freshwater systems yielded similar rates

(Gargetal., 2011a). Themechanismof abioticphotochemical O

2production from NOM has usually been represented by a reactionin which NOM is photoexcited to a triplet state, which subse-quently transfers an electron to O2to yield O

2 and an oxidized

organic moiety (Cooper et al.,1988,1989):

NOM + hv+ O2 NOM+

+ O2 (2)

The pathway(s) by which this overall reaction occurs are still notcertain, although the details are slowly being unraveled. Whileit was thought that photoexcited NOM may eject free aquated

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electrons, this now appears unlikely (Thomas-Smith and Blough,2001). Instead, the photoexcited organic moiety can undergo arange of possible energetic and redox transitions, particularly inhumic and fulvic substances that possess a wide range of func-tional groups and redox potentials. The mechanism of organicradical formation in photoexcited NOM was first studied in detailbyBlough (1988)but, despite considerable advances over the last

20 years, the precise details of O

2 formation still remain elusive.The mechanisms were thought to be similar to those involvedin O2 production from photoexcited quinones, which have beenstudied extensively (e.g.,Garget al., 2007b and references therein),however the situation in humic and fulvic-type NOM is compli-cated considerably by theexistence of multiple chromophores(DelVecchio and Blough,2002,2004;Boyle et al.,2009) and redox-active moieties (Aeschbacher et al.,2010), many of which maybe linked under exposure to light through charge-transfer com-plexes (Del Vecchio and Blough,2004). More recently,Garg et al.(2011b)proposed a mechanism in which quinone-like moietieswithinNOM are reduced by some otherelectron donor within theNOM structure under solar radiation, with the reduced organic

moiety then reacting with dissolved oxygen (in either the tripletor singlet state) to yield O2 :

NOM + hv organicelectrondonor NOM (3)

NOM + O2 NOM + O2 (4)

The overall process is complicated considerably by formationof a range of other radical sinks for O 2 , but the net result is thatNOM is oxidized by O2 to yield O

2 through the involvement

of several moieties within the NOM, some of which may act asphotocatalysts.

Consistent with the concept of labile transfer of electrons

between the range of LRACs present in oxygenated waters, a sec-ond indirect mechanism of photochemical superoxide formationinvolves formation of reduced metals via ligand-to-metal charge-transfer reactions, with subsequent oxygenation of the reducedmetal. The role of such reactions involving ironorganic com-plexes in marine systems has been recently reviewed byBarbeau(2006), and similar reactions can also occur with complexes ofcopper (Jones et al.,1985).

BIOLOGICALPRODUCTION

Biological production of O2 in the extracellular milieu has beenreported from culture studies involving a wide range of environ-mentally occurring aquatic microorganisms, including eukaryotic

microalgae from the class Raphidophyceae (Oda et al., 1997;Mar-shall et al.,2002,2005;Yamasaki et al.,2004;Garg et al.,2007a);dinoflagellates and prymnesiophytes(Yamasaki et al.,2004;Mar-shall et al.,2005); marine diatoms from the genus Thalassiosira(Kustka et al.,2005); marine cyanobacteria from the generaSyne-chococcus (Rose et al.,2008b), Lyngbya (Rose et al.,2005) andTrichodesmium (Godrant et al.,2009); a marine alphaproteobac-terium from the genus Roseobacter (Learman et al., 2011); thefreshwater cyanobacterium Microcystis aeruginosa (Fujii et al.,2011); the unicellular protozoan coral symbiont Symbiodinium(Saragosti et al., 2010); fungi and yeasts including Aspergillus

nidulans (Lara-Ortz et al., 2003) and Saccharomyces cerevisiae(Shatwell et al., 1996), as reviewed byAguirre et al.(2005); and theheterotrophic bacteria Paracoccus denitrificans (Henry and Vig-nais, 1980) and Escherichia coli (Korshunov and Imlay, 2006).Rates of ESP vary enormously between these different organisms,with the maximum reported rates being from the marine Raphi-dophyceanChattonella marinaof up to 5pmolcell1 h1 (Oda

et al.,1997;Garg et al.,2007a). Additionally, recent field studieshave provided evidence from several marine environments sug-gesting that a significant proportion of ESP occurs via biologicallymediated processes, with measured rates of biological ESP up to1 n M h1 in the Great Barrier Reef lagoon(Rose et al.,2010) andup to 20nMh1 in the Gulf of Alaska (Hansard et al., 2010).These studies have predominantly involved either field studies inmarine environments, or culture studies of organisms that typi-cally inhabit marine environments, with notable exceptions beingthe studies involvingM. aeruginosa,P. denitrificans(which is typ-ically rather ubiquitous in soils),A. nidulans,S. cerevisiae, andE.coli. While there is still insufficient evidence to make definitivegeneralizations, the occurrence of these processes across such a

wide range of microorganisms in the marine environment, cou-pled with evidence for ESP by a range of terrestrial multicellularorganisms such as lichens (Beckett et al.,2003) and plants (Bol-well, 1999), suggests that microbial ESP probably occurs to at leastsome extent in the vast majority of surface waters.

Biological production of O2 in the intracellular environmenthas been studied for several decades and many of the processesinvolved are well understood. In eukaryotic organisms, intracel-lular O2 production is believed to be highly compartmentalized,with the majority occurring via reduction of O2inside the mito-chondria and through the action of NADPH oxidase (NOX)enzymes, which are typically located inside vesicles (Auchre andRusnak,2002). In photosynthetic eukaryotes, O2 is also pro-

duced in significant amounts in the chloroplasts (Lesser,2006).In prokaryotic organisms, which lack intracellular compartmentsseparated by cellular membranes,the majority of intracellular O2production is thought to occur during O2 reduction by the res-piratory electron transport chain (Auchre and Rusnak, 2002),which is also used for electron transport during photosynthesis inphotosynthetic prokaryotes such as cyanobacteria (Lesser,2006).It is extremely unlikely that such intracellular processes can bethe direct source of ESP because the polar O2 anion is unable toreadily diffuse through the lipid bilayer that isolates the cytoplasmfrom the external milieu (Korshunov and Imlay,2002). HOO

can diffuse at a rate approaching that of water (permeability ofP= 9 104 cm s1 = 3.2 102 m h1; Korshunov and Imlay,

2002), but as HOO

is only a small proportion of O

2 aroundneutral pH, this flux is limited. The flux of O2 out of the cell isgiven by:

Flux= HOO PAO2

(5)

WhereHOOis thefraction of O2 that isin the HOO

form, Pisthe permeability of HOO,Ais the cell surface area, and [O2 ]is the difference between the intracellular and extracellular O2concentrations. In a typicalhealthycell, the steady-state intracel-lular O2 concentration is around 100 pM(Imlay and Fridovich,

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1991). Considering an exemplary spherical cell with a diameter of20m, and assuming that the steady-state extracellular O2 con-centration 100pM, the calculated flux is 8 zmol cell h1. Evenif we assume that the cell is suffering from severe oxidative stress,resulting in an intracellular O2 concentration of 10 nM, the fluxis still only 800 zmol cell h1. This is several orders of magnitudeless thanmeasured rates of ESP from the range of aquatic microor-

ganisms surveyed to date. Additionally, O

2 concentrations in theexternal milieu may be similar to, if not greater than, intracel-lular O2 concentrations, such that diffusion of O

2 across the

lipid bilayer, even if it could occur at substantial rates, would infact occur into the cell under these conditions by virtue of theconcentration gradient.

Another possible source of ESP is release of intracellular O2during cell lysis. Assuming that the rate of cell lysis = rate of celldivision to maintain a steady-state population, with spherical cells20mindiameterundersevereoxidativestresssuchthattheintra-cellular O2 concentration wasagain 10 nM,then even in a rapidlygrowing population with a division rate of 1 h1 the rate of ESP bythis pathway would be only 4 zmol cell h1. Of course more rapid

lysis could temporarily result in a more rapid release of intracellu-lar O2 by this pathway, but such rates could not be sustained toyield the steady-state concentrations of O2 that have been mea-sured in natural waters to date. Thus the majority of O2 foundin the extracellular milieu of aquatic microorganisms must, ingeneral, be produced extracellularly.

While mechanisms of biological ESP have been reasonably wellstudied in human cells, mechanisms of biological ESP in aquaticmicroorganisms are generally not well understood. Enzymes fromthe NOX and dual oxidase (DUOX) families located at the cyto-plasmic membrane are known to be responsible for ESP in themajority of human cells, notablyincluding phagocytes responsiblefor the respiratory burst immunological defense phenomenon

(Vignais, 2002; Lambeth, 2004). Cellsurface located NOX enzymeshave also been shown to be responsible for ESP inC. marina(Kimet al.,2000), the fungus A. nidulans(Lara-Ortz et al.,2003) andnumerous other fungi and yeasts (Aguirre et al.,2005), certainplant cells (Torres et al., 1998;Sagi and Fluhr, 2001), and the coralStylophora pistillataand its symbiont Symbiodiniumsp. (Saragostiet al.,2010). Addition of diphenyleneiodonium chloride (DPI),which is a specific inhibitor of NOX enzymes, has also been shownto dramatically inhibit ESP in cultures of the diatoms Thalassiosiraweissflogiiand T. pseudonana(Kustka et al., 2005). BLAST searchesof sequenced genomes reveal the presence of genes encoding forpolypeptide sequences similar to those found in NOX proteins inthese twodiatoms (Kustka et al., 2005)andinawiderangeof other

eukaryotic aquatic microorganisms, however this does not neces-sarily indicate the occurrence of ESP mediated by NOX enzymesin these organisms, since NOX enzymes are also found intracel-lularly as discussed previously. BLAST searches of the genomes ofprokaryotic aquatic microorganisms reveal that such genes are notalways present, suggesting that ESP by prokaryotes may occur viaone or more other mechanisms.

Finally, it is worth reiterating that it is often difficult to establishwhich species is reduced first during ESP; in the case of biologicalESP, it is not always clear whether O2 is reduced to O

2 directly

by enzymes, or whether O2 is produced through oxygenation of

some other reduced LRAC formed directly by enzymatic reduc-tion. In the case of E. coli, O2 production in the organismsperiplasm has been inferred to occur through the oxidation ofsoluble, reduced quinones, with electrons originating from trans-fer across the cytoplasmic membrane via the respiratory electrontransport chain (Korshunov and Imlay, 2006). The cell surface fer-ric reductase system of the yeastS. cerevisiaepossesses similarities

to the NOX system at the genetic level (Shatwell et al.,1996), andis capable of reducing a range of LRACs (Kosman, 2003) includingO2to yield O

2 (Lesuisse et al., 1996). The biogeochemical signif-

icance (if any) of which LRAC is reduced first is not yet certain,however it is clear that caution should be used when assigninga particular mechanism for cell surface reductase activity on thebasis of genetic homologyor biochemical studies conductedundera limited set of chemical conditions.

CHEMISTRY OF REACTIONS BETWEEN SUPEROXIDE AND

IRON

OVERVIEW

In aqueous solutions, O2 can react in three major ways (Sawyer

and Gibian,1979;Sawyer and Valentine,1981;Sawyer,1991):

1. As a reductant. The O2 anion is a mild reducing agent capa-ble of directly donating electrons via an outer-sphere processto suitable electron acceptors (e.g., transition metals and theircomplexes).

2. As an oxidant. HOO is capable of oxidizing suitable electrondonors via direct electron transfer, but this is highly unfavor-able for the O2 anion due to the extreme instability of the

O22 anion in aqueous solution at most pH values. However,O2 is capable of indirectly inducing oxidation by either hydro-gen atom or proton extraction from suitable substrates, withsubsequent reactions resulting in their net oxidation.

3. As a Lewis base (electron pair donor). The O

2 anion maybe coordinated in the inner sphere of various metal ions inaqueous solution, and can also participate in radicalradicalcoupling with a range of organic radical cations.

The role of O2 as a Lewis base is restricted in natural aquaticenvironments because the maximum concentration of O2 thatis attainable is typically too low (due to its consumption via otherreactions) in comparison to other species to have any significanteffect. In practice, therefore, O2 behaves primarily as a reductantor oxidant in natural waters. Early laboratory studies of reactionsbetween aqueous Fe and O2 typically used pulse radiolysis togenerate O2 and UVvisible spectrophotometry to observe the

subsequent reactions (Jayson et al.,1969,1973;Buettner et al.,1983;Bull et al.,1983;Rush and Bielski,1985). While useful inunderstanding the chemistry of Fe and O2 , results from thesestudies must be applied to environmental systems with cautionbecause the use of high concentrations of both Fe and O2 couldfavor reactions that are not important at lower, environmentallyrelevant concentrations. This is particularly true for oxidation ofFe(II) by O2 , where the initial step appears to typically involvecoordination of O2 in the inner sphere of Fe(II). Furthermore,natural systems involve a host of other competing reactions dueto the range of other species present.

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The potential effect of O2 on Fe in environmental systemswas first specifically studied byVoelker and Sedlak (1995). Theyconcluded that O2 functioned primarily as a reductant of Fe(III)over the pH range 58 (where O2 dominates over HOO

), andthat substantial amounts of Fe(II) could be expected to be pro-duced in certain environmental systems as a result of this process.More recent studies have typically used lower concentrations of Fe

and O

2 that are more representative of what is typically foundin natural systems, largely due to the possibilities afforded by thedevelopment of new techniques such as the MCLA chemilumines-cence method, and have considered a range of different conditionsthat are more relevant to natural waters. Fe species can be broadlyclassified in such systems into three main groups:

1. Mononuclear inorganic complexes, which include hydrolysisspecies and a range of relatively weak complexes with small,commonlyoccurringinorganicligandssuchaschloride,sulfate,bromide, fluoride, carbonate, and phosphate. These complexesinterconvert rapidly compared to the other reactions we willconsider, and may thus be assumed to be at equilibrium at all

times.2. Mononuclear organic complexes, which occur with a widerange of naturally occurring organic ligands such as humic andfulvic-type materials, polysaccharides, siderophores, and otherbiologically produced compounds.

3. Polynuclear complexes, which include amorphous oxyhydrox-ide solids, various iron minerals, and aggregates containingvarying proportions of iron, organic material, and potentiallyother species.

Within each of these classes, individual Fe atoms may be presentin either the Fe(II) or Fe(III) redox state, and the transformationsbetween these redox states may be mediated by O 2 (Figure 2).

THERMODYNAMICS

Due to pKaof O2/HOO

being in the environmentally relevantrange, and HOO being considerably more oxidizing than O2,the standard redox potential of the O2/O

2 couple can vary con-

siderably from +120 mV at pH 4.8 to 160 mV at pH 4.8(Sawyer,1991). In the majority of natural surface waters, whichare in the neutral to slightly alkaline pH range, the standardredox potential of the O2/O

2 couple is thus 160 mV. There-

fore, under standard conditions at around neutral pH, O 2 isa mild reducing agent. In contrast, the standard redox poten-tials of various Fe(II)/Fe(III) couples vary from +1000mV depending on pH and complex speciation (Pierre

and Fontecave, 1999; Pierre et al., 2002). Despite the overlapbetween the standard redox potentials of the O2/O

2 couple and

those of many Fe(II)/Fe(III) species redox couples, an impor-tant caveat when considering the feasibility of redox reactionsis that these redox potentials apply to standard conditions, i.e.,[O2] = [O

2 ] = [Fe(II)] species = [Fe(III)] species. As discussed

in detail byPierre et al. (2002), such conditions are irrelevantin most natural systems. Under conditions typical for a neu-tral air-saturated surface water (pH 7 and [O2] = 250 M), theactual redox potential of the O2/O

2 couple is +335mV when

[O2 ] = 1 pM and +158mV when [O2 ] = 1 nM. An equally

FIGURE 2 | Transformations between various pools of Fe as mediated

by O

2 . (A) Polynuclear complexes, which contain multiple Fe atoms and

may also contain other inorganic and organic ligands.(B)Mononuclear

inorganic complexes.(C)Mononuclear organic complexes. Fe(III) in all

pools may be reduced by the O

2 anion to Fe(II), while Fe(II) in all pools may

be oxidized to Fe(III) primarily by HOO, but also indirectly by O

2 . The dotted

arrows denote the dissociative reduction (DR) pathway, while the dashedarrows denote the non-dissociative reduction (NDR) pathway for Fe(III)

reduction by O

2 .

important consideration for the thermodynamic reducibility ofa particular form of Fe by O2 is the concentration of relevantFe(II) and Fe(III) species. The reduction of an organic Fe com-plex [denoted as Fe(II)L in the reduced state and Fe(III)L in theoxidized state] is thermodynamically feasible if:

log([Fe (II) L]/[Fe (III) L]) 10 pM can increase [Fe

]T(=[Fe(II)] + [Fe(III)]), provided that the dominant form(s) ofFe(II) (e.g., organic Fe(II) complexes) are relatively labile com-pared to the dominant form(s) of Fe(III). In a spatially homoge-neous system, it is unimportant whether cell surface ferrireduc-tases reduce Fe(III)to Fe(II) that then reacts with O2toyield O

2 ,

or whether cell surface oxygen reductases reduce O2to O2 that



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then reacts with Fe(III) to yield Fe(II), unless ferrireductases passFe(II) directly to the uptake site without release of free Fe(II).In such a system, direct reduction of O2 by cells maybe moreeffective in increasing Fe bioavailability, since rates of O2 reduc-tion by an oxygen reductase may be faster than rates of Fe(III)reduction by a ferrireductase simply because the concentrationof O2 is much greater than that of Fe(III) in most oxygenated

environments. However in a spatially heterogeneous system, aferrireductase mechanism would likely be more efficient due tobiological compartmentalization and diffusion resulting in higherconcentrations of Fe(II) near the site of cellular uptake comparedto in thebulk solution.Understandingthe chemistry of Feand O2at a detailed mechanistic level, and a more rigorous understand-ing of the role of physical transport processes, is needed to fullyassess the potential role of O2 in increasing Fe bioavailability

in a range of aquatic environments. On the basis of the infor-mation presently available, however, it seems that extracellularO2 has the potential to significantly increase Fe bioavailabilityunder some conditions at least and may therefore be an impor-tant part of the complicated process of Fe acquisition by aquaticorganisms.

ACKNOWLEDGMENTSI wish to acknowledge the contributions over the past decade ofmy close colleague Prof. T. David Waite and his research group atthe University of New South Wales to the work that underpinnedthe development of many of the ideas discussed in this paper. Ialso gratefully acknowledge funding from the Australian ResearchCouncil through projects DP0987188 and DP0987351 that partlysupported the writing of this paper.

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