SA1 Oxygen Binding Heme Proteins

8P Research Symposium transcriptional and post-translational responses to hypoxia are SA1 not fully understood. Our model proposes that mitochondria function as cellular oxygen sensors through a process that Oxygen binding heme proteins involves interaction of molecular oxygen with Complex III of M.T. Wilson the electron transport chain. Hypoxia results in a paradoxical increase in the release of reactive oxygen species from the outer Biological Sciences, University of Essex, Colchester, UK surface of the inner mitochondrial membrane. These signaling Myoglobin (Mb) and Hemoglobin (Hb), the respiratory pig- levels of oxidant stress lead to the activation of transcription fac- ments of mammals and some molluscs, annelids and arthro- tors including Hypoxia-Inducible Factor (HIF-1 and HIF-2) and pods, belong to an ancient super-family of heme associated glo- NF-kB, and they trigger cell-specific post-translational responses bin proteins. Members of this family share common structural to hypoxia. Stabilization of HIF-α protein in hypoxia is abro- and spectral features. They also share some general functional gated when genetic modifications to the electron transport chain characteristics such as the ability to bind ligands, e.g. O2, CO and lead to loss-of-function in terms of the ability to generate ROS NO, at the iron atom and to undergo redox changes. These prop- signals during hypoxia. Conversely, genetic modifications to erties are used in vivo to perform a wide range of biochemical Complex II that induce a chronic increase in mitochondrial ROS and physiological roles. production lead to the stabilization of HIF-α under normoxic While it is acknowledged that the major role of Hb is to bind conditions. In tumor cells, this gain-of-function leads to an oxygen reversibly and deliver this to the tissues, this is not the increase in tumor cell growth in tissue culture and in vivo, which sole function of the protein. In addition, the often-stated role of is mediated by the increase in HIF activation. These findings sug- Mb as an oxygen storage protein is possibly a misconception. gest that Complex III plays a dual role in the cell, through its Both Hb and Mb may, for example, express enzymic activities involvement in energy transduction and in the detection of cel- that are important to their function e.g. NO oxidase activity or lular hypoxia. peroxidatic activities that may be partially responsible for patho- Authors have confirmed where relevant, that experiments on physiology following haemorrhage. animals and man were conducted in accordance with national The biochemical and biophysical evidence for these functions and/or local ethical requirements. will be described and the discussion extended to include proteins that have been discovered comparatively recently and that are expressed at low levels within the cell, e.g. the neuroglobins and cytoglobins. These proteins are wide spread throughout the SA3 animal and plant kingdoms and may have specialist roles in oxygen delivery to particular sites within the cell but may also per- Potassium channel protein partners: gas sensing in the form roles associated with O sensing and signalling and in the 2 cardiovascular system organism’s response to stress e.g. by providing protection from reactive oxygen species. Similarly, hemoglobins are widespread P.J. Kemp 1, S.P. Brazier1, N. Baban1, D. Riccardi1, C.T. Müller1, in plants and bacteria and may serve similar protective functions. C. Peers2 and S.E. Williams1 The talk will present the essential features shared by these pro- 1School of Biosciences, Cardiff University, Cardiff, UK and teins and discuss how these are tuned in different organisms to 2Cardiovascular Research Institute, University of Leeds, Leeds, UK accomplish an extensive range of physiological tasks. The ability to react rapidly to dynamic changes in arterial blood Support from the BBSRC and Wellcome Trust is gratefully gas composition is crucial for optimal delivery of molecular oxy- acknowledged. gen (O2) to respiring tissues. The principal sensory components Authors have confirmed where relevant, that experiments on of this homeostatic mechanism are the carotid bodies. Ideally animals and man were conducted in accordance with national situated in the bifurcation of the common carotid artery, they and/or local ethical requirements. respond muliplicatively to hypoxia, hypercapnia, pH and hypo- glycaemia.When activated, secretion of a variety of transmitters by the carotid body glomus cells results in augmented input to the respiratory centres of the brain stem. Thus, during reduced SA2 O2 availability, activation of the carotid bodies promotes increased rate and depth of ventilation as a compensatory Oxygen sensing by the mitochondrial electron transport response to systemic hypoxia. At the cellular level, hypoxia pro- chain: Role of reactive oxygen species in signal transduction motes inhibition of plasma membrane potassium channels of in hypoxia carotid body glomus cells which leads to calcium influx and P.T. Schumacker transmitter release. In rat glomus cells, two potassium channels have been implicated in the hypoxia-dependent depolarization Department of Pediatrics, Northwestern University, Chicago, IL, USA - a specific member of the tandem P-domain potassium chan- Oxygen sensing is a fundamental biological process that is nel family (almost certainly the TASK sub-type (Buckler et al., required for development, for successful transition from pla- 2000) and the calcium-activated, large conductance potassium cental to lung respiration at birth, for normal oxygen homeostasis channel (BKCa - (Peers, 1990)). It appears likely that both BKCa throughout life, and for tumor angiogenesis and progression at and the TASK-like potassium channel contribute to carotid body the end of life. Despite the importance of this process in health O2 sensing but, until recently, the identity of the O2 sensor had and disease, the molecular mechanisms by which cells trigger remained elusive. Copyright 2007 The Biochemical Society, the British Pharmacological Society and The Physiological Society Research Symposium 9P In the search for a potential O2 sensing mechanism which could Authors have confirmed where relevant, that experiments on account for rapid and reversible inhibition of BKCa channels, we animals and man were conducted in accordance with national carried out a proteomic screen for potential protein partners of and/or local ethical requirements. α the BKCa -subunit using immunoprecipitation, two-dimen- sional electrophoresis and mass spectrometry. Of particular note was the protein partnership (verified by double-label immuno- α SA4 cytochemistry) of BK with an O2-dependent enzyme, hemeoxy- genase-2 (HO-2). In the presence of O2 and NADPH, this enzyme Oxygen sensing by protein hydroxylases oxidizes cellular heme to generate carbon monoxide (CO), iron and biliverdin. Downstream products of HO-dependent heme P. Ratcliffe catalysis have been reported to play important roles in a wide Nuffield Department of Medicine, University of Oxford, Oxford, variety of biological tissues including the immune, the central Europe, UK nervous and the cardiovascular systems. Of particular interest Recent work has defined novel oxygen sensitive pathways that is the observation that HO-2 is expressed in the carotid body and signal hypoxia by modulating post-translational amino acid that CO has a major impact on carotid body chemotransduc- hydroxylation at specific sites. Hypoxia inducible factor (HIF) tion (Prabhakar et al., 1995). is an alpha/beta heterodimeric transcriptional complex that plays Todefine the molecular mechanism linking HO-2 activity to chan- a key role in directing cellular responses to hypoxia. The activ- nel inhibition during hypoxia, we have employed single channel ity of HIF is itself controlled by post-translational hydroxylation studies to show that BKCa channel (expressed in both HEK293 cells at prolyl and asparaginyl residues within the alpha-sub-units. and natively in carotid body glomus cells) activity is robustly and HIF prolyl hydroxylation governs proteolytic regulation of HIF reversibly activated by the downstream products of HO-2,biliverdin whereas HIF asparaginyl hydroxylation (FIH) modulates inter- and CO (the latter via addition of the chemical CO-donor, action with transcriptional co-activators. These hydroxylations [Ru(CO)3Cl2]). In the presence of O2, addition the HO-2 co-sub- are catalysed by a set of non-haem Fe(II) 2-oxoglutarate (2OG) strates, heme and NADPH, evoke an increase in channel activity. dependent dioxygenases. During catalysis, the splitting of molec- Importantly,in their continued presence,hypoxia evokes a depres- ular oxygen is coupled to the hydroxylation of HIF and the oxida- sion in channel activity which is much larger than that observed in tive decarboxylation of 2-oxoglutarate to give succinate and CO2. the absence of HO-2 co-substrates.These observations suggest that Hydroxylation at two prolyl residues within the central ‘degra- HO-2 enzymatic activity confers a significant enhancement to the dation domain’ of HIF-alpha increases the affinity for the von O2 sensing ability of the HO-2/BKCa protein complex. In support Hippel-Lindau (pVHL) E3 ligase complex by at least three orders of this notion,selective knock-down of HO-2 protein by RNA inter- of magnitude, thus directing HIF-alpha polypeptides for prote- ference dramatically depresses tonic channel activity and the olytic destruction by the ubiquitin/proteasome pathway. Since

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