Vol. 483: 1–17, 2013 MARINE ECOLOGY PROGRESS SERIES Published May 30 doi: 10.3354/meps10367 Mar Ecol Prog Ser FREE ACCESS FEATURE ARTICLE Heme b in marine phytoplankton and particulate material from the North Atlantic Ocean David J. Honey1, Martha Gledhill1,*, Thomas S. Bibby1, François-Eric Legiret1, Nicola J. Pratt1, Anna E. Hickman1, Tracy Lawson2, Eric P. Achterberg1 1Ocean and Earth Science, University of Southampton, National Oceanography Centre-Southampton, Southampton SO14 3ZH, UK 2Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK ABSTRACT: Concentrations of heme b, the iron- containing prosthetic group of many hemoproteins, were measured in 6 species of marine phytoplankton (Dunaliella tertiolecta, Emiliania huxleyi, Thalassio - sira weissflogii, T. oceanica, Phaeodactylum tricor- nutum and Synechococcus sp. WH7803) that were subjected to variations in iron concentration. Changes in heme b in response to reduced light and nitrate were also ex amined for E. huxleyi and T. oceanica. Results from laboratory cultures were compared with heme b determined in particulate material in the North Atlantic. In cultures, heme b made up 18 ± 14% (SE) of the total iron pool. Reduced iron and nitrate concentrations resulted in a decreased intracellular heme b concentration, expressed as per mole carbon. Chlorophyll a (chl a) to heme b ratios in E. huxleyi and In heme b, the central iron atom (orange) is bound to four D. tertiolecta in creased in response to limited light nitrogen atoms (blue) of a tetrapyrrole ring composed of carbon (grey), hydrogen (white) and oxygen (red) atoms. and nutrient availability, but slightly decreased or did not change in the diatoms and the cyanophyte Syne- Image: M. Gledhill and T. Bibby chococcus sp. WH7803. The heme b:particulate or- ganic carbon (POC) and chl a:heme b ratios in the North Atlantic were within the range observed in phytoplankton cultures. In the surface mixed layer, INTRODUCTION decreases in heme b:POC ratios were linked to de- creases in nutrient concentrations. Chl a:heme b Iron is among Earth’s most abundant elements; ratios increased with depth and were thus primarily however, iron concentrations can be extremely low affected by light availability. Relative relationships (<0.2 nmol l−1) in oceanic surface waters (De Baar & between heme b, chl a and POC in the North Atlantic De Jong 2001). Iron is involved in many fundamental likely represented a change in the ability of cells to biological processes, including photosynthesis, respi- undertake cellular processes driven by chl (light a ration, nitrogen fixation and nitrate reduction (Gei- harvesting) and heme b (e.g. electron transport) ac- der & LaRoche 1994, Sunda 2012). Hemoproteins cording to ambient light and nutrient conditions. form an important reservoir of iron in oceanic micro- KEY WORDS: Hemoprotein · Iron · Electron transport · organisms and are 1 of 3 types of iron-containing Cytochrome · Nutrient limitation · Celtic Sea proteins, along with iron sulphur and iron oxygen proteins (Frausto da Silva & Williams 2001). Hemo- Resale or republication not permitted without written consent of the publisher proteins are a functionally diverse group involved *Corresponding author. Email: [email protected] © Inter-Research 2013 · www.int-res.com 2 Mar Ecol Prog Ser 483: 1–17, 2013 in electron transfer, substrate oxidation and oxygen bins, catalases, peroxidases, cytochrome P450 and transport, storage and reduction (Chapman et al. b-type cytochromes (Mochizuki et al. 2010). Hemes 1997). In particular, hemoproteins facilitate the 2 are closely related to the photosynthetic pigments most fundamental processes in biology: respiratory chlorophyll and phycobilin, which are produced and photosynthetic electron transfer via b- and c- via the same tetrapyrrole biosynthetic pathway as type cytochromes (Mochizuki et al. 2010). However, heme (Cornah et al. 2003, Mochizuki et al. 2010). knowledge of the abundance of hemoproteins in During tetrapyrrole biosynthesis, iron or magnesium phytoplankton and their distribution in the marine is chelated into protoporphyrin IX to produce heme environment is currently very limited (Gledhill 2007, or chlorophyll, respectively. A complex feedback Saito et al. 2011), although it has been shown that mechanism determines the allocation of protopor- low iron concentrations result in reduced abundance phyrin IX to either the heme or chlorophyll branch of of other iron proteins in phytoplankton (Bibby et al. the synthesis pathway (Vavilin & Vermaas 2002). 2009, Marchetti et al. 2009, Saito et al. 2011, Richier The aim of our study was to investigate heme b in et al. 2012). phytoplankton and particulate material in the ocean. Hemoproteins contain heme(s) (iron-porphyrin The abundance of heme b and its response to complexes) as the prosthetic group. Hemes act as a changes in iron, nitrate and light conditions was direct source of iron for some marine heterotrophic explored in 6 species of phytoplankton originating bacteria (Hopkinson et al. 2008), and heme uptake from the North Atlantic. Species from different phy- genes have been identified in diverse regions of the logenetic groups were chosen to allow comparison ocean (Desai et al. 2012, Hopkinson & Barbeau 2012). with previous published work on the abundance of Hemes are highly toxic to cells when not associated heme b-containing proteins (e.g. Greene et al. 1991, with proteins, meaning heme abundance is tightly 1992, Strzepek & Harrison 2004, Suggett et al. 2007, linked to hemoprotein abundance (Espinas et al. 2012). Allen et al. 2008). Relationships between the concen- The many functions attributed to hemoproteins arise tration of heme b, chlorophyll a (chl a), particulate from variations in iron ligation and charge state, as organic carbon (POC) and particulate organic nitro- well as the addition of different substituents on the gen (PON) were investigated. In addition, particulate tetrapyrrole ring (Frausto da Silva & Williams 2001). concentrations of heme b were determined in sam- Four specific heme structures are relatively common ples collected from the Celtic Sea, part of the north- in organisms (hemes a, b, c and d) of which heme b west European Shelf Sea, and from a cross-sectional (iron protoporphyrin IX) is the most widespread (sub-)tropical North Atlantic transect (NA transect) (Espinas et al. 2012). Heme b is associated with glo- close to the 24th parallel (Fig. 1). Fig. 1. Stations sampled in the Celtic Sea (inset) and the route of the North Atlantic transect (NA transect; red line). Five oceanographic regions of the NA transect were defined as the Florida Straits (FS), Gulf Stream (GS), West Tropical North Atlantic Gyre (WTNAG), East Tropical North Atlantic Gyre (ETNAG) and Azores Current (AC). Colours represent bathymetry (blue: >2000 m; green: 250–2000 m; grey: <250 m) Honey et al.: Heme b in marine systems 3 MATERIALS AND METHODS runs. As cultures reached the end of the exponential phase, phytoplankton were transferred to fresh me - Phytoplankton growth conditions and sampling dium to obtain an initial biovolume close to 1.5 × 106 µm3 l−1. Dunaliella tertiolecta, Thalassiosira Batch cultures of Dunaliella tertiolecta Butcher weiss flogii, T. oceanica, Emiliania huxleyi and Phaeo- (1959) (PCC83, CCAP 19/6B), Emiliania huxleyi B11 dactylum tricornutum were grown at 19°C under cool (CCAP 920/8), Thalassiosira weissflogii (Grunow in white fluorescent lights at 150 µmol quanta m−2 s−1 van Heurck) Fryxell & Hasle (1977) (PCC541, CCAP (high light) or 45 µmol quanta m−2 s−1 (low light) on a 1085/1), T. oceanica (Hustedt) Hasle et Heimdal 12:12 h light:dark cycle. Synechococcus sp. WH7803 (PCC692, originally CCMP1005) and Phaeodactylum was grown at 22°C under 30 μmol quanta m−2 s−1 light tricornutum Bohlin 1897 (PCC670, CCMP 632) were on a 12:12 h light:dark cycle (Wilson et al. 1996). grown in an experimental medium prepared from Cell density and biovolume were monitored daily filtered seawater (0.2 µm, Sartobran, Sartorius) col- on fresh culture samples with a Beckman Coulter lected from the North Atlantic Subtropical Gyre counter (Multisizer 3, Meritics) using 3 mol l−1 NaCl (24.1−29.5° N and 23.4−27.6° W, January 2005). Basal solution as diluent and electrolyte. The maximum −1 nutrient concentrations were 0.15 ± 0.04 µmol l quantum yield of PSII (Fv/Fm) of dark-adapted cells nitrate, 0.06 ± 0.06 µmol l−1 phosphate, 0.5 ± 0.03 µmol were determined in the mid-exponential phase l−1 silicate and 0.54 ± 0.05 nmol l−1 dissolved iron using a FASTtracka II Fast Repetition Rate fluoro - (errors are expressed as SD, unless otherwise stated). meter (Chelsea Technologies; Kolber et al. 1998, All chemicals and consumables were purchased from Ross et al. 2008). Cultures were filtered onto glass Thermo Fisher Scientific unless otherwise stated. microfibre filters (MF300, nominal pore size 0.7 µm, The culture medium was enriched with 150 (high) or Fisher Scientific) for the determination of heme b 15 (low) µmol l−1 nitrate, 10 µmol l−1 phosphate, (10 to 200 ml), the photosynthetic protein PsbA 100 µmol l−1 silicate, 100 µmol l−1 ethylenediamine (D1 protein of PSII: 50 to 200 ml), chl a (10 to 30 ml) tetraacetic acid (EDTA), 50 nmol l−1 cobalt, 100 nmol and POC and PON (hereafter POC/N: 10 to 200 ml) l−1 molybdenum, 20 nmol l−1 copper, 115 nmol l−1 towards or at the end of the exponential phase. manganese, 80 nmol l−1 zinc, 10 nmol l−1 selenium Filters for POC/N were ashed (450°C, 16 h) prior to and f/2 vitamins. Iron (Fe) was added separately from use. Filters for subsequent heme b and PsbA ana - a stock solution of 4.5 mmol l−1 FeEDTA at concen - lysis were stored at −80°C, whilst filters for chl a trations between 0.5 nmol l−1 and 1.5 µmol l−1 added and POC/N were stored at −20°C.