Th The en enzymatic basis of f en energy-generation Lecture 3: - PowerPoint PPT Presentation

Th The en enzymatic basis of f en energy-generation Lecture 3: Respiration of inorganic compounds Dr r Chris Greening Lecturer / Group Leader Monash University May 6 6 2016 2016

Lecture 3: Respiration of inorganic compounds I. I. Prokary ryotic ic versatil ilit ity II. I. Nitr itrif ific icatio ion / / denit itrif ific icatio ion III II. Aerobic ic H 2 respir iratio ion IV. Anaerobic ic H 2 respir iratio ion

Prokaryotes inhabit every environment  Prokaryotes (bacteria, archaea) are present in large cell numbers in every environment on earth: from animal guts to hot springs to deep-sea sediments. They can flourish in such environments because of their metabolic flexibility. Three I’m currently studying: Rob obin inson Rid Ridge Mari ariner Tren ench Moun ount Ngauruhoe (Antarctic (A ic de desert rt) (de (deep-sea ven ents) (v (vol olcanic ic cr crater) r)

Mitochondria are efficient but inflexible  Mitochondria have very limited flexibility. Their e - donors are all derived from organic carbon compounds and their sole e - acceptor is O 2 . Energy is transduced through highly efficient but inflexible linear electron transport chains.  In many ecosystems, the available electron donors (organic carbon sources), acceptors (O 2 ), and physical conditions are insufficient to sustain animal life.

Prokaryotes are highly metabolically diverse  Microorganisms can prosper in almost all ecosystems due to their respiratory flexibility: They can substitute organic e - donors for inorganic e - donors (e.g. H 2 ) - They can substitute O 2 for anaerobic e - acceptors (e.g. NO 3 2- ) - - Employ branched electron transport chains that can use multiple donors and acceptors  In addition to respiration, most microorganisms can sustain energy-conservation by fermentation (substrate-level phosphorylation) in the absence of exogenous e - acceptors. Many organisms also capture light and/or fix inorganic carbon.

Alternative e - sources and sinks E o ’ Red edox cou ouple CO 2 / CO -0.492 V CO 2 / Formate -0.432 V 2H + / H 2 -0.414 V CO 2 / CH 4 -0.239 V 2- / H 2 S SO 4 -0.218 V CoM-S-S-CoB / -0.140 V CoM-SH + CoB-SH Fumarate / +0.030 V Succinate - / NH 3 NO 2 +0.340 V - / NO 2 - NO 3 +0.431 V Fe 3+ / Fe 2+ +0.770 V

Alternative e - sources and sinks E o ’ Red edox cou ouple Prim rimary deh ehydrogenase CO 2 / CO Carbon monoxide dehydrogenase -0.492 V CO 2 / Formate Formate dehydrogenase -0.432 V 2H + / H 2 Hydrogenotrophic hydrogenase -0.414 V CO 2 / CH 4 Methane monooxygenase -0.239 V 2- / H 2 S SO 4 -0.218 V Various inc. sulfide oxidoreductase CoM-S-S-CoB / N/A -0.140 V CoM-SH + CoB-SH Fumarate / +0.030 V Succinate dehydrogenase Succinate - / NH 3 NO 2 Ammonia monooxygenase +0.340 V - / NO 2 - NO 3 +0.431 V Nitrite oxidoreductase Fe 3+ / Fe 2+ +0.770 V Iron oxidase

Alternative e - sources and sinks E o ’ Red edox cou ouple Prim rimary deh ehydrogenase Ter erminal red eductase CO 2 / CO Carbon monoxide dehydrogenase N/A -0.492 V CO 2 / Formate Formate dehydrogenase N/A -0.432 V 2H + / H 2 Hydrogenotrophic hydrogenase Hydrogenogenic hydrogenase -0.414 V CO 2 / CH 4 Methane monooxygenase Methanogenesis pathways -0.239 V 2- / H 2 S SO 4 -0.218 V Various inc. sulfide oxidoreductase Various inc. sulfite reductase CoM-S-S-CoB / N/A Heterodisulfide reductase -0.140 V CoM-SH + CoB-SH Fumarate / +0.030 V Succinate dehydrogenase Fumarate reductase Succinate - / NH 3 NO 2 Ammonia monooxygenase Nitrite reductase +0.340 V - / NO 2 - NO 3 +0.431 V Nitrite oxidoreductase Nitrate reductase Fe 3+ / Fe 2+ +0.770 V Iron oxidase Iron reductase

Directionality depends on environment  Microorganisms can effectively mix-and-match the electron donors and acceptors they use depending on what is available in the environment. e - don e - acce Ter erm onors cceptor Aerobic organotrophy Organic: sugars, amino acids, formate, O 2 methane, acetylene, lignin, TNT, etc. - , Fe 2+ , etc. Aerobic lithotrophy Inorganic: H 2 , CO, NH 3 , NO 2 O 2 - , NO 2 - , SO 4 2- , Fe 3+ , CO 2 , fumarate, H + , Anaerobic organotrophy Organic: sugars, amino acids, formate, NO 3 methane, etc. etc. - , NO 2 - , SO 4 2- , Fe 3+ , CO 2 , etc. Anaerobic lithotrophy Inorganic: H 2 , CO, H 2 S, etc. NO 3  In oxic environments, a wide range of compounds can be used as fuel sources for aerobic respiration (e.g. NH 3 , H 2 , H 2 S, CH 4 ). In anoxic environments, the same compounds can be produced as electron sinks during anaerobic respiration. All down to simple energetics.

If there’s a negative Δ G...  A golden rule in microbial energetics is that, if an e - donor and an e - acceptor are available for a thermodynamically-favourable reaction to occur, some organism will be able to mediate it. This is even the case when the free energy released is very low. Some examples: E o ’ Δ E o ’ Δ G o ’ Process Half Ha lf-equations NAD + + H + + 2e - → NADH Aerobic -0.320 V -219 kJ mol -1 +1.136 V ½ O 2 + 2H + + 2e -  H 2 O organotrophy +0.816 V Fe 3+ + 1e -  Fe 2+ Aerobic +0.770 V +0.046 V -8.8 kJ mol -1 ½ O 2 + 2H + + 2e -  H 2 O lithotrophy +0.816 V CO 2 + H + + 2e -  Formate Anaerobic -0.432 V -3.5 kJ mol -1 +0.018 V 2H + + 2e -  H 2 organotrophy -0.414 V 2H + + 2e -  H 2 Anaerobic -0.414 V -163 kJ mol -1 +0.845 V - + 2H + + 2e -  NO 2 - + H 2 O lithotrophy NO 3 +0.431 V  The approaches used greatly vary both within and between organisms. Some prokaryotes have specialist metabolism that enables them to dominate certain niches, whereas others are highly versatile and can adapt to a wide range of environments.

The redox tower of e - acceptor utilisation  In environments where there is more than one e - acceptor available (e.g. O 2 , NO 3 - ), the highest potential acceptor (O 2 ) will be used over the others (NO 3 - ). The lowest potential e - acceptors (i.e. protons) are only used in the most energy-poor environments.

Main factors shaping e - acceptor utilisation Regulation: Competition: Metabolically flexible organisms (e.g. Metabolically inflexible organisms reliant on low-potential e - acceptors (e.g. obligate facultative aerobes such as E. coli ) sense e - acceptor availability. If multiple acceptors anaerobes such as sulfate-reducers) are are available, they upregulate the outcompeted in energy-rich environments. reductases of high-energy acceptors (e.g. Their ETCs yield less energy per organic molecule oxidised (lower H + /2e - ratios) than cytochrome c oxidase) and downregulate the others (e.g. nitrate reductase). e.g. E. coli . They in turn grow much slower.

Lecture 3: Respiration of inorganic compounds I. I. Prokary ryotic ic versatil ilit ity II. I. Nitr itrif ific icatio ion / / denit itrif ific icatio ion III II. Aerobic ic H 2 respir iratio ion IV. Anaerobic ic H 2 respir iratio ion

Nitrogen cycle

Nitrification: N compounds as e - donors  Nitrification is a two-step process which oxidizes ammonia and nitrite as fuel sources. Due to high potential of the e - donors, O 2 is required as an e - acceptor. - + 2 H 2 O + 2 H + 2 NH 3 + 3 O 2  2 NO 2 Δ E o ’ = +0.816 - +0.340 = + 0.476 V - + O 2  2 NO 3 - Δ E o ’ = +0.816 - +0.431 = + 0.385 V 2 NO 2  Despite the biogeochemical significance of nitrification, only a few organisms can mediate the process. Those that do are specialist lithotrophs that grow on few other fuel sources.  Classically thought different organisms living in symbiosis mediate NH 3 oxidation (e.g. Nitrososphaera ) and nitrite oxidation (e.g. Nitrobacter ). However, complete nitrifiers (e.g. Nitrospira ) were recently identified through high-throughput sequencing (Nature 2015). Sergei Ser i Wino nogr gradsky (18 (1856 – 1953): ): Di Discoverer of of lith thotrop ophy and and ni nitr trif ific ication

Electron transport chains in nitrification  Specialised primary dehydrogenases, ammonia monooxygenase and nitrite oxidoreductase (structures not solved), input electrons into ETC. Electrons transferred via cytochrome c to proton-translocating cytochrome c oxidase. Δ p drives F 1 F o ATP synthase. Ammon onia ia oxi xidatio ion Nitr trit ite oxid xidatio ion e.g. e. g. Nitr itrososphaera, , Nitr itrospira e. e.g. g. Nitr itrobacter, , Nitr itrospira

Reversed electron flow in nitrification  All organisms generate reductant (e.g. NADH) to sustain biosynthetic processes. Oxidation of organic compounds, H 2 , and CO can be favourably coupled to NAD + reduction. However, it is - oxidation to NAD + reduction. thermodynamically impossible to couple NH 3 and NO 2  Reversed electron flow is the solution. Δ p can be consumed to drive a reversed endergonic e - transfer pathway: nitrite  cytochrome c  Complex III  UQ  Complex I  NAD + .