Metalloproteins in the Biology of Heterocysts

Abstract

Cyanobacteria are photoautotrophic microorganisms present in almost all ecologically niches on Earth. They exist as single-cell or filamentous forms and the latter often contain specialized cells for N₂ fixation known as heterocysts. Heterocysts arise from photosynthetic active vegetative cells by multiple morphological and physiological rearrangements including the absence of O₂ evolution and CO₂ fixation. The key function of this cell type is carried out by the metalloprotein complex known as nitrogenase. Additionally, many other important processes in heterocysts also depend on metalloproteins. This leads to a high metal demand exceeding the one of other bacteria in content and concentration during heterocyst development and in mature heterocysts. This review provides an overview on the current knowledge of the transition metals and metalloproteins required by heterocysts in heterocyst-forming cyanobacteria. It discusses the molecular, physiological, and physicochemical properties of metalloproteins involved in N₂ fixation, H₂ metabolism, electron transport chains, oxidative stress management, storage, energy metabolism, and metabolic networks in the diazotrophic filament. This provides a detailed and comprehensive picture on the heterocyst demands for Fe, Cu, Mo, Ni, Mn, V, and Zn as cofactors for metalloproteins and highlights the importance of such metalloproteins for the biology of cyanobacterial heterocysts.

Keywords: Nostocales; bioenergetics; cyanobacteria; electron transport chains; heterocysts; metabolism; metalloenzymes; metalloproteins; metals; oxidative stress.

Figure 1

Biologically relevant metals found in organisms. Life is primarily based on the six bulk elements H, C, N, O, P, and S (blue), which are involved in basic biological organic chemistry. Alkali metals are highlighted in yellow, alkaline earth metals are shown in orange, and d-block metals, which include transition metals and group 12 metals (Zn and Cd), are presented in light red. f-block elements are not shown but their positions are indicated for lanthanides (₅₇La*) and actinides (₈₉Ac**).

Figure 2

Representative metal clusters in heterocyst metalloproteins. (A) P cluster (top) and FeMo-co (middle; Mo nitrogenase, 4wza), and FeV-co (bottom; V nitrogenase, 5n6y). (B) Ni–Fe (top) and [3Fe–4S] (bottom) clusters (Ni–Fe hydrogenase, 3rgw). (C) [4Fe–4S] cluster F_B (photosystem I, 6hqb). (D) Rieske cofactor (cytochrome b₆f, 4ogq). (E) Cu center (plastocyanin, 2cj3). (F), [2Fe–2S] cluster (ferredoxin FdxH, 1frd). (G) Cu–Cu center Cu_A (cytochrome c oxidase, 1qle). (H) Fe–Fe center (flavodiiron protein, 1ycf). (I) Mn center (Mn SOD, 1gv3). (J) reduced (left) and oxidized (right) Fe–Fe center (rubrerythrin, 1lko/1lkm). (K) Fe–Fe ferroxidase center (Dps protein, 1n1q). (L) [Fe–4S] cluster (rubrerythrin, 1lko). (M) Zn centers bound to the substrate analogue phosphoglycolohydroxamate (PGH; fructose-1,6-bisphosphate aldolase, 1b57). (N) catalytic [4Fe–4S] cluster bound to isocitrate (aconitase, 1b0j). h, homocitrate.

Figure 3

Metal requirements in the photosynthetic electron transport chain in heterocysts. Electrons can be transferred via three PSI-dependent (photosystem I-dependent) routes during photosynthesis. They can flow cyclically between the cytochrome b₆f complex (Cyt b₆f) and PSI via the soluble electron carriers plastocyanin (PC) or cytochrome c₆ (Cyt c₆) and the enzyme FNR (ferredoxin:NADP(H) oxidoreductase), which enable a flux of electrons from PSI-reduced ferredoxin (Fd) and Cyt b₆f. Electrons can also be transferred linearly from the respiratory NDH-1 (type-I NAD(P)H dehydrogenase) complex to Cyt b₆f via the plastoquinone (PQ) pool, before being transferred to PSI via PC or cytochrome c₆. Fd is the final electron acceptor in the photosynthetic electron transport chain and is used as an electron donor for the nitrogenase or to form NADPH via FNR. Another cyclic route connects PSI and the NDH-1 complex via Fd. All cyclic and linear photosynthetic electron transport chains create a proton gradient across the membrane that is used by ATP synthase to produce ATP. Subunits of the NDH-1 complex, cytochrome b₆f and PSI are not shown for the sake of simplicity.

Figure 4

Metal requirements in the respiratory electron transport chain in heterocysts. During respiration, electrons are transferred from the NDH-1 complex to the cytochrome b₆f complex (Cyt b₆f) through the plastoquinone (PQ) pool, before being shuttled to the cytochrome c oxidase (Cox) via plastocyanin (PC) or cytochrome c₆. In heterocysts, the alternative respiratory terminal oxidases quinol oxidase (Qox) and cytochrome bd quinol oxidase (Cyd) accept electrons from PQH₂ (plastoquinol) and reduce O₂. The respiratory electron transport chain generates a proton gradient across the membrane that is used by ATP synthase to produce ATP. Subunits of the NDH-1 complex, cytochrome b₆f, and respiratory terminal oxidases are not shown for the sake of simplicity.

Figure 5

Main energy metabolism and carbon/nitrogen fluxes in heterocysts. The OPPP (oxidative pentose phosphate pathway) and the Krebs cycle are the major routes used by heterocysts to generate reducing equivalents, while glycolysis has a smaller contribution. Metalloenzymes (red circles) and enzymes catalyzing reactions that generate (yellow circles) or require (green circles) NAD(P)H are indicated. Abbreviations for metabolites: Suc, sucrose; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6P, fructose 1,6-bisphosphate; G3P, glyceraldehyde 3-phopshate; PEP, phosphoenolpyruvate; Ac-coA, acetyl-coA; Mal, malate; OA, oxaloacetate; Cit, citrate; Isocit, isocitrate; 2-OG, 2-oxoglutarate. Abbreviations for enzymes: G6PD, glucose 6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; FBA, class-II fructose bisphosphate aldolase; FBP, fructose bisphosphatase; G3PD, glyceraldehyde 3-phosphate dehydrogenase; PK, pyruvate kinase; PEPS, PEP synthase; PDH, pyruvate dehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase; MDH, malate dehydrogenase; CS, citrate synthase; AcnB, aconitase, IDH, isocitrate dehydrogenase; GS, glutamine synthetase. Cyanophycin metabolism and the reported transfer to vegetative cells of the cyanophycin-derived dipeptide β-aspartyl-arginine [120] are not included in the scheme.