Heme-based sensors in biological systems

158

Heme-based

sensors in biological

systems

Kenton R Rodgers The past

several

years

have

been

witness

of advancement in the understanding respond to changes in the availability that are toxic and/or presently constitute sense

NO,

0,

and CO

and CooA,

The major approaches They

and

has grown

advances have to elucidation

include

rate

crucial to survival. Heme-based sensors the majority of the proteins known to to initiate

the chemistry

adapt to changes in their availabilities. characterized members of this class, FixL

to a staggering

of how organisms of diatomic molecules

growth

substantially resulted of both

during

the past

from a broad function and

in the understanding

between the heme and well as alternate means

required

to

Knowledge of the three soluble guanylate cyclase, year.

range of mechanism.

of the interplay

protein in soluble for its stimulation.

guanylate cyclase, as Insight into the O,-

induced structural changes in FixL has been supplied by the single crystal structure of the heme domain of Bradyrhizobium japonicum.

Finally,

the ligation

interchange

that facilitates

established

by spectroscopic

Address Department of Chemistry, Fargo, ND 58105-5516, Current

Opinion

environment

CO

and

Biology

by CooA

mutagenesis

Ladd Hall, North USA

in Chemical

and ligand

sensing

Dakota

1999,

has been techniques.

State

University,

Soluble 3:158-i

67

http://biomednet.com/elecref/1367593100300158 0 Elsevier

Science

Abbreviations BJFiXL 6JFixLt-t cGMP ESR GSH GSNO GTP Hb HO HS ImH ls Mb NOS PAS P(O*) RmFixL RmFixLN/RmFixLH RmFixL*/RmFixLT rR sGC SOD

Ltd ISSN

bound ligand. Studies of biological signaling in prokaryotic and eukaryotic organisms has revealed a new class of heme-containing proteins, the heme-based sensors. The function of these proteins is to detect the presence of NO, 0, or CO by their coordination to the heme and to initiate chemistry that culminates in the organism’s response to changes in availability of the ligand. To date, there are only three such proteins known and characterized, the mammalian NO receptor cyclase soluble guanylate cyclase (sGC), the bacterial 0, sensor-kinase FixL, and the bacterial CO sensor CooA. These proteins each comprise a heme-containing sensor domain that converts ligand-binding free energy into protein conformational free energy, which is transmitted to another domain where it initiates substrate turnover or DNA binding. Recent work in this field has significantly advanced the molecular-level understanding of the interplay between coordination chemistry of the heme centers and their immediate protein environments. In the following review, I have summarized advances in the knowledge of each of the aforementioned heme-based sensor proteins during the past several years.

1367-5931

Bradyrhizobium japonicum FixL Heme domain from B. japonicum FixL cyclic 3’,5’-guanosine monophosphate electron spin resonance glutathione .S-nitrosoglutathione 5’-guanosine triphosphate hemoglobin heme oxygenase high-spin imidazole low-spin myoglobin nitric oxide synthase domains containing two direct sequence repeats (S, and S2 boxes) of -50 residues partial pressure of 0, Rhizobium meliloti FixL heme domain from R. meliloti FixL heme kinase from R. meldoti FixL resonance Raman soluble guanylate/guanylyl cyclase superoxide dismutase

each

Introduction Heme proteins and enzymes constitute a large class of biological macromolecules exhibiting a broad spectrum of functions. These include selective ligand binding for the purposes of storage, transport or catalytic activation of the

guanylate

cyclase

NO serves in a number of biological capacities. It is produced by NO synthases (NOSs) upon their activation by Ca2+bound calmodulin. It has been shown to serve as a primary intercellular signaling molecule critical to vascular smooth muscle relaxation, neurotransmission and inhibition of platelet aggregation. NO is also generated in large quantities by macrophage NOS as a cytotoxin in the course of the immune response to invading organisms. The target for NO is sGC, a heterodimeric enzyme comprising an a subunit of 73-88 kDa and a heme-containing p subunit of 70 kDa [l--5,6’]. The ferrous b-type heme is bound to the protein through the imidazole (ImH) sidechain of His105 [Z-4]. Notably, the Fe-ImH bond in ferrous sGC is the weakest of all known histidine-bound heme proteins, as shown by its 204 cm-l Fe-ImH stretching (vFe-rmH) frequency [7,8,9”]. When NO binds to the heme as a distal axial ligand, the weak Fe-ImH bond is cleaved to yield a live-coordinate low-spin (LS) ferrous nitrosyl heme [5,6’,7,8,9”,10,11]. Protein conformational reorganization caused by rupture of the Fe-ImH bond is thought to be responsible for the up to 400-fold increase in sGC activity toward conversion of 5’-guanosine triphosphate (GTP) to the intracellular second messenger, cyclic 3’,5’-guanosine monophosphate (cGMP). Hence, the chemistry of NO with sGC is the lynchpin of the aforementioned cellular responses to endogenous NO-based signals. Several aspects of sGC function as a heme-based receptor are not understood. Specifically, the means by which NO is transported to the receptor is not understood, the mechanism of NO release from activated sGC is unclear, and the putative stimulation of sGC by endogenous CO produced

Heme-based

Figure

sensors

in biological

systems

Rodgers

159

1

The fate of NO in signaling pathways. Reaction of L-arginine and 0, to generate NO and O,‘is catalyzed by NOS. Reaction of the two products yields peroxynitrite (ONOO-). Less than 1% of GSH reacts with ONOOand the reaction is inhibited by CO, In the presence of SOD, Os- is scavenged, leaving diffusible NO to activate sGC. In the presence of GSH, 25-450~ of total NO/O,is converted to GSNO. The mechanism for conversion of GSNO back into NO may involve multiple mechanisms. The resulting NO is available for sGC activation. Adapted from [12’].

Synaptic transmission L-arginine ’

o’,

\

~

NO

J ONOO-

and transport

of nitric

oxide

Being an odd-electron molecule, NO is prone to facile reactions with 02-, which is co-produced with NO by NOS [12’]. This rapid scavenging reaction yields the peroxynitrite ion (ONOO-) [13,14], which does not directly stimulate sGC but may play a role in cytotoxicity of NO produced in macrophages. (For a review on ONOO-, see Groves’ paper [Z&-235] in this issue.) On this basis, the effective in viva lifetime of NO should be short. This expectation has been substantiated by increased sGC activation in the presence calmodulin-activated NOS and superoxide dismutase (SOD) as a competitive O,- scavenger [12’]. Thus intercellular transport of NO probably requires a chaperone that can either protect NO by scavenging O,-, or use O,- to generate a less reactive compound capable of escorting the NO moiety to its target. NO and O,- have been shown to react with glutathione (GSH) to give the thionitrite, S-nitrosoglutathione (GSNO) [15]. This compound has a long lifetime under physiological conditions and releases NO in the presence of trace Cu+ [l&16’]. The ready availability of GSH (-5 mM in mammalian tissues) and accessibility of GSNO make it a prime candidate for an NO storage sink and escort. Observation of this chemistry in w&o has led to the proposal that trace copper or an as yet undiscovered enzyme could present NO to sGC via catalytic decomposition of GSNO. The flow chart in Figure 1 illustrates this GSH-based model of NO transport and release. Hemoglobin (Hb) has also been proposed as a reversible in r&o NO sink that participates in regulation of blood flow

-jyrP

GTP

GSH A

Cu+ or enzymes

w GSNO

*

I NO

I Neuroprotection No storage

I Neurotoxicity Tissue injury

Stabilization

2;

+ 0,’

co2

in &o by heme oxygenase (HO) remains to be clarified. Recent work by a number of groups has defined and shed light on these interesting issues.

NO

[ 17-191. Upon exposure to NO, R-state human Hb (relaxed conformation, high 0, affinity) in the form of HbA-CO undergoes site-specific S-nitrosylation at Cys93 to yield S-nitroso HbA-NO. The crystal structure of this form of Hb has been solved at 1.8 A resolution, confirming the formation of S-nitroso HbA-NO [ZO”]. In the current model for blood flow regulation by Hb, release of 0, from the heme and NO from Cys93 are cooperatively coupled to induce vasodilation and increased blood flow to 02deprived tissues [17-19,20”]. In effect, the S-nitroso group may behave as a weak allosteric T-state (tense conformation, low 0, affinity) effector which, upon release, also increases blood flow through sGC-mediated vasodilation.

Inactivation

of soluble

guanylate

cyclase

In spite of the stability of sGC-NO, the rate constant for NO release is second only to T-state Hb-NO, which also contains five-coordinate nitrosyl hemes. On this basis, it has been suggested that when endogenous NO levels drop, NO is simply released spontaneously under the auspices of LeChatlier’s principle. Half times for spontaneous NO release in viva have been estimated at between 35 s and 2 min, suggesting that spontaneous release of NO from stimulated sGC could play a role in down-regulation of cGMP [21,22’]. The possibility of a natural effector able to accelerate NO release prompted a resonance Raman (rR) study of the sGC heme in the presence of Mgz+ and either GTP or cGMP [9”]. These experiments revealed that both product (cGMP) and substrate (GTP) cause the appearance of a second vNO Raman band in the presence of Mgz+. The authors suggest that cGMP binds in or near the distal heme pocket and perturbs the protein-NO interactions by increasing distal hydrophobicity. It was hypothesized that

160

Bio-inorganic

Figure

2

chemistry

YC-1. (a) The two-dimensional wireframe structure. (b) A stereoview of an AM-1 energy-minimized structure. (Energy minimization was carried out by the author using the Chem3D module of ChemOffice.)

(b)

Current

binding of accumulated cGh4P to the heme pocket in oivo could be a feedback signal that decreases the heme affinity for NO by driving the dissociation of NO and the consequent down-regulation of cGMP production. Whether such a feedback-driven mechanism for NO release is thermodynamically and kinetically reasonable awaits thorough studies of&,, (i.e. binding rate constant) and R,rr(i.e. release rate constant) values for NO in the presence of cGMI? Another model for deactivation is based on the sensitivity of ferrous nitrosyl hemes to oxidation. In the presence of O,, the ferrous nitrosyl heme is oxidized to the unactivated ferric sGC nitrate complex [23’]. Alternatively, oxidation of sGC-NO by a one-electron oxidant such as (Fe[CN]# yields the corresponding labile ferric heme-NO complex [23’]. In fact, saliva from the Amazonian bloodsucking bug R/zoa’nius prolixw contains a ferric heme that binds NO reversibly so it can be delivered to its host where it affects vasodilation and inhibits platelet aggregation during feeding [24,25]. Hence, there is firm biological precedence for labile ferric heme-NO complexes, From a chemical standpoint, this is an attractive model, since it provides for active deactivation of sGC-NO; however, the notion of redox-driven binding and release of NO would gain substantial credence upon identification of a physiological one-electron oxidant and a complimentary reductant.

Opimon

in Chemical

Carbon soluble

Bdogy

monoxide guanylate

as a possible cyclase

activator

of

Like NO, CO binds strongly to heme iron centers, is cytotoxic and has one endogenous source in vertebrate animals. CO is produced along with billiverdin in the catalytic oxidation of heme by the enzyme HO. A constitutive form of HO (HO-Z) has been shown to co-localize with sGC in brain tissues, suggesting the possibility of CO involvement in neurotransmission [26,27]. Moreover, i?~vitro tJC labeling experiments have revealed that 14C0 evolution from neuronstracks the accumulation of cGMP [28]. CO hasalso been shown to elicit in U~VOand in vitro vascular [29’] and intestinal [30”] smooth muscle relaxation, providing substantial and compelling evidence for NO-like physiological responsesto CO. However, the current understanding of the sGC stimulation mechanism is at odds with the cause and effect evidence for CO-induced cGMP accumulation. Although CO binds tightly to the heme of sGC to form a LS ferrous complex, the CO adduct is six-coordinate [11,31,32]. With its proximal Fe-ImH bond intact, sGC-CO exhibits cyclase activity a mere two to six times the basal,in vitro level 131,321.It should be noted that the HO and NOS inhibitory drugs, tin protoporphyrin IX and zinc protoporphyrin IX, respectively, are used in studies of the coupling between HO-derived CO and the physiological effects of cGh4P.These inhibitors have been shown to

Heme-based

Figure

sensors

in biological

systems

Rodgers

161

3

A schematic illustration of the role of FixL in the signaling pathway for transcriptional activation of nifA and fixK. When the heme in FixL binds O,, the heme is LS and kinase activity is inhibited. When 0, is removed from the heme iron, the Fe(ll) is HS and the kinase is activated. The phospho-FixL that is generated provides a phosphoryl group for the generation of of phospho-FixJ. Phospho-Fix-l is the active form of the transcriptional activator. Phospho-FixL-O2 has been shown to have phosphatase activity towards phospho-Fid. The membrane-bound domain has been omitted from the phospho-FixL for clarity. Adapted from [44,50].

[L-

A

Kinase

]

PO,

I

Fix L

affect the activity of sGC directly [33’]. Hence, interpretation of the results from such studies should include consideration of these effects. Recent studies have shown that in the absence of NO, sGC activity can be increased over basal levels by about tenfold upon treatment with the fury1 benzylindazole, YC-1. Figure 2 shows a wireframe structure and stereographic projections of an AM-1 energy-minimized YC-1 structure. (AM-1 is a semi-empiral energy calculation.) Insensitivity of the W-visible spectra to YC-1 suggests that the drug does not directly interact with the heme. Rather, it appears to act as an allosteric effector [34-36,37”,38”]. Interestingly, CO and YC-1 together stimulate sGC activity to the same extent as NO, suggesting the possibility of one or more natural endogenous effecters capable of amplifying the small stimulating effect of CO alone. If a natural effector analogous to YC-1 exists, knowledge of the YC-1 shape will be useful in identification of effector candidates. Such a molecule would have to be present either in the proximicy of HO and sGC or be co-produced with CO. Among the candidates that might be worthy of investigation are the pyrrolic products of HO-catalyzed heme degradation.

FixL FixL, an 0, sensor, and FixJ, its response regulator, belong to a large family of bacterial two-component signal transduction systems [39]. Together they control transcription of at least two genes required for nitrogen fixation in symbiotic R&izobia [40-X]. FixL contains a histidine kinase domain whose activity is responsive to the spin state of a b-type heme which resides in its Oz-sensing domain [43]. Cinder anaerobic conditions, such as occurs in root nodules of leguminous plants, the heme is largely high-spin (HS) Fe(II) and its kinase domain is active.

Figure 3 illustrates the known roles of FixL in the FixL/J signal transduction pathway. In a reaction with ATP, the kinase domain undergoes autophosphorylation at a conserved histidine residue. Transfer of the phosphoryl group to FixJ renders it transcriptionally active, causing up regulation of nifA and.fixK. IJnder aerobic conditions, the heme is LS with O2 bound, kinase activity is inhibited, and FixJ is not transcriptionally activated. FixL also exhibits phosphatase activity towards phospho-FixJ [44]. Hence, up- and down-regulation of nifA and fixK transcription are likely to be steep in time, as FixL autonomously regulates both accumulation and depletion of phospho-FixJ. Recent work on these proteins has been aimed at structure determination and elucidation of the mechanismby which changesin heme ligation and spin state are transmitted to the kinase domain to initiate the chemistry of cellular response to changesin the partial pressureof 0, [P(O,)]. These studies have examined a number of deletion derivatives of membrane-bound Rliixobirm rnek&~’ FixL (RmFixL) and the soluble Bradyrhizobium japonicum FixL (BlFixL) proteins. These proteins include RmFixLN [43]/RmFixLH [45], and RtnFixL* [43]/RmFixLT [45], which correspond to slightly different truncations of the heme domain and functional heme kinases, respectively. BlFixL and its heme domain BjFixLH have alsobeen studied [4.5,46”].

Heme

coordination

chemistry

Although the kinasedomain of FixL is homologouswith the histidine kinases of two-component sensors[39], the O,sensingheme domainsshow no homology with other known heme proteins [45,46”]. The W-visible absorbancesignatures of numerous FixL heme ligand complexes [43], heme binding studieswith site-directed histidine mutants [47], and the electron spin resonance (ESR) spectra of RmFixLN-NO, RmFixL-NO [48] and RmFixLT-I%0 1491

162

Bio-inorganic

chemistry

indicate that the proximal heme ligand in RmFixL is a histidine residue. The presence of a proximal histidine in B’FixL was recently confirmed by X-ray crystallography [46”]. UV-visible spectra of ferric FixLs indicate that the native heme pockets are hydrophobic and cannot stabilize the coordination of water to the heme iron [4.5]. rR spectra show the hemes in ferric RmFixLN and RmFixL* to be five-coordinate and HS [SO]. Both NMR and ESR spectra are also consistent with the S = 5/Z ferric HS hemes in RmFixL and BjFixL derivatives [50,51’,52’,53]. The recent crystal structure of ferric BjFixLH confirms the presence of a five-coordinate ferric heme in the isolated heme domain [46”]. The affinity of FixLs for O,, CO and NO is generally lower than for myoglobin (Mb) and Hb [45,49]. The decreased affinity appears to be primarily due to smaller Ron values for FixL. This has been attributed to a decrease in the reactivity of the heme-iron in FixL relative to Mb. It can be rationalized in terms of the heme-iron being more strongly confined to the proximal side of the heme because of protein conformational tension applied through the proximal Fe-ImH bond [48,SO]. This model is based on the low vFGIrnr~ frequency (2 11 cm-r) in deoxyRmFixLN and deoxyRmFixL*, consistent with a bond weakened by conformational tension [54’]. The Fe-O, bond strength in RmFixL-O2 is similar to that in Mb-O,, insofar as it is reflected in the vFe-02 frequency (-571 cm-l) 1481. The steric factors influencing ligand-binding rate to RmFixLH have been probed using exogenous ImH [49,55’]. These results suggest that the heme is readily accessible by the large polar ImH ligand. In an effort to detect subtle perturbations of the heme pocket by interactions between the heme and kinase domains of FixL, distribution of heme species at alkaline pH has been investigated [Sl’,S2’,56]. Based on spectroscopic [Sl’,W] and activity [S6] data, the relationship between kinase activity and protonation state of the protein may implicate the most acidic heme-domain tyrosine residue in stabilizing the protein conformation required for signal transmission.

Residues

required

for heme

binding

A mutagenesis study has been carried out to determine which of the 18 invariant hydrophobic residues are required for assembly of holoFixL [57]. It was determined that uptake of heme by FixL requires histidine in the proximal ligand position. Although a number of conclusions regarding structural roles and location of these hydrophobic amino acids with respect to the heme were drawn, the validity of some of them has been called into question by the recent &FixLH crystal structures. However, increases reported in the autoxidation rates for IleZ09-+His, IleZlO+His and IleZlO+Ala are elucidating. As the BjFixLH crystal strrtctures show, both of these residues cover entryways into the distal heme pocket. Increased rates of ferrous heme autoxidation upon replacement of these bulky hydrophobic

‘gatekeeper’ sidechains with either more polar or smaller sidechains are consistent with increased accessibility of the heme by water [S8]. Even though the distal pocket is lined with hydrophobic residues and the ferric FixL-OH2 adduct is not stable, water does appear to interact with the ferrous heme. Accessibility of the ferrous heme by water, even in the wild type protein, is also supported by an 0, protection effect on the FixL autoxidation rate constant [S&59].

Heme

domain

structure

Crystal structures of ferric F’FixLH and &FixLH-CN were recently reported to 2.4 A and 2.7 A, respectively. These structures provide fascinating insight into the possible mechanisms for signal initiation by the heme domain of FixL, and they are the first structures of a sensor domain containing both the S, and S, boxes (i.e. the conserved regions) of the PAS sequence [46”]. It was predicted that the S motif, present in S, and S,, comprises mainly B topology with a central helix. Also, secondary structure composition for RmFixLN was predicted with some accuracy on the basis of circular dichroism spectra [SO]. The BjFixLH structure confirms this prediction and, together with sequence homology data for a large number of sensor domains [60’], suggests that the PAS fold is likely to be ubiquitous in sensor domains of the plant and animal kingdoms. The heme-binding domain of FixL is one of the PAS domains that harbors a cofactor, There are two other O,sensing PAS domains from NifL and Aer, which contain flavin adenine dinucleotide and sense P(0,) by change in oxidation state of the flavin. The photoactive yellow protein (PYP) exhibits PAS homology and contains a 4-hydroxycinnamyl chromophore covalently bound to the protein through a thioester linkage to a cysteine sidechain [61’,62’]. Changes in tertiary structures of FixL and PYP upon heme ligation [46”] and photoexcitation [62’], respectively, suggest that the loops flanking the central helix are key to regulation. Sidechains in these regions of the structures exhibit the largest conformational rearrangement in response to their respective stimuli and probably contribute to the conformational coordinate along which the signals are transmitted to their target domains. In FixL, conformational rearrangement of the loop between the carboxyl end of the central helix and the amino boundary of the S2 box appears to respond to flattening of the heme, which occurs upon formation of the LS ferric B’FixLH-CN [46”]. Since only the heme domain structure is presently known, it is not clear whether the signal pathway out of the heme domain leads directly to its kinase domain or to that of a partner in a dimeric complex. A thorough understanding of the signaiing mechanism of the FixL/J regulatory system will require studies to address the question of ligand-coupled dimerization.

CooA The photosynthetic bacterium Rhdospidhm rubrum can grow using the energy evolved from oxidation of CO to

Heme-based

Figure

sensors

in biological

systems

Rodgers

163

4

-l

Fe(lll)

Fe(l I)

Fe(l I )-CO Current

The heme ligation states CooA is six-coordinate provided by the protein and a histidine residue. residue remains bound

of CooA in various forms. In its ferric form, with the heme axial coordination being amino acid residue sidechains of a cysteine In the ferrous form, the axial histidine to the iron. The state of the cysteine ligation

CO, as its sole energy source. CO oxidation is coupled to HZ evolution by a multicomponent CO oxidation system that is induced in the presence of CO [63,64]. It has been shown by mutagenesis that CooA is necessary for COdependent expression of the two operons that encode the enzyme system for CO oxidation [65”]. CooA is a 24.6 kDa protein comprising a sensor domain, which contains the first 131 amino acid residues, and a DNA-binding domain [66]. It is a member of the family of single-component regulators, which includes the fumarate nitrate reduction and CAMP receptor proteins [67]. These proteins bind to specific DNA sequences upon uptake of effector molecules or upon reaction with 0,. Early studies of CooA suggested that its effector was CO [68]. This suggestion was later strengthened by the finding that the sensor domain of CooA contains a b-type heme that can be interconverted between ferrous, ferric and ferrous-CO forms, all of which exist as LS complexes [69]. Figure 4 illustrates the interconversion of these forms of CooA. ESR [70”] and mutagenesis [71”] studies have identified the links between the heme and protein as Cys75 and

Opinion

in Chemical

Biology

is not as clear; the Fe-cysteine bond appears to be significantly weakened due to protonation, or broken. When CO is coordinated to ferrous CooA, a six-coordinated complex is observed with a histidine and CO as the axial heme ligands. This illustration was constructed on the basis of observations reported in [70”,71”].

His77. Both axial coordination sites of the heme are occupied by these sidechain ligands to give a LS ferric heme. In the ferrous form, the heme ligation is unclear; W-visible data suggest that CooA is either six-coordinate with a weakly bound and perhaps protonated cysteine sidechain or that the cysteine is not coordinated to the heme iron. Treatment of ferrous CooA with CO yields a LS six-coordinate CO complex with a Soret (So-&,, K-X*) maximum at 422 nm. Since LS six-coordinate ferrous hemes with thiolate axial ligands are characterized by B-band maxima near 4.50 nm, the 422 nm maximum indicates that the thiolate of Cys75 is displaced by CO to give a LS six-coordinate CO-histidine complex [70”,71”]. CO rebinding monitored at 420 nm subsequent to CooA-CO photolysis indicates that there are three kinetic phases for bimolecular reassociation of CO with the heme [72’]. These phases are postulated to represent CO binding to three intermediates along the ligation-induced conformational transition coordinate. Treatment of CooA-CO with target DNA selectively diminished the rate constant for the fast rebinding phase. The same

164

Bio-inorganic

chemistry

report included heme rR spectra of CooA-CO in the presence and absence of target DNA that reveal population of a distinct heme conformer in the DNA-bound protein. The frequency of the vFewCO band was shifted up by 33 cm-t in the DNA-bound state, which was interpreted as indicative of an increase in interaction between the distal heme pocket and CO in the DNA-bound protein. This interpretation is consistent with the observed decrease in the rate of the fast CO rebinding reaction. These experiments provide compelling kinetic and spectroscopic evidence that the heme- and DNA-binding regions of the protein are energetically coupled.

An O2 sensor for the mammalian hypoxic response Maintenance of oxygen homeostasis in mammalian organisms depends upon multiple responses to hypoxia [73]. Current evidence strongly suggests that the sensor responsible for regulating transcription of erythropoietin (Epo), vascular endothelial growth factor, tyrosine hydroxylase, inducible NOS and others is a heme oxidase protein [73-75,76’,77-79,80’.,81’,82]. Since Epo is expressed in response to diminished P(O,), it has been adopted as a physiological paradigm for the hypoxic response [76’,77-79,8O”,Sl’,S2,83’,84,85]. The potential impact of identifying the O2 sensor and attaining a molecular-level understanding of its function make this an exciting area to follow in the near future.

5.

Zhao Y, Schelvis JPM, Babcock GT, Marletta MA: Identification histidine 105 in the pl subunit of soluble guanylate cyclase the heme proximal ligand. Biochemisfry 1998, 37:4502-4509.

of as

6. .

Dierks EA, Hu S, Vogel KM, Yu AE, Spiro TG, Burstyn JN: Demonstration of the role of scission of the proximal histidineiron bond in the activation of soluble guanylyl cyclase through metalloporphyrin substitution studies. I Am Chem Sot 1997, 119:7316-7323. This study clearly demonstrates the requirement for exogenous ligand-coupled reversibility of proximal Fe-histidine bond formation. Conclusions are based on resonance Raman studies of soluble guanylyl cyclase reconstituted with various metalloporphyrins exhibiting different axial coordination chemistry. 7.

Zhao Y, Hoganson C, Babcock GT, Marletta MA: Structural changes in the heme proximal pocket induced by nitric oxide binding to soluble guanylate cyclase. Biochemistry 1998, 37:12458-l 2464.

8.

Denium G, Stone JR, Babcock GT, Marletta MA: Binding of nitric oxide and carbon monoxide to soluble ouanvlate cvclase as observed by resonance Raman specbokopy. Biochemistry 1996, 35:1540-l 547.

9. *

Tomita T, Ogura T, Tsuyama S, lmai I, Kitagawa T: Effects of GTP on bound nitrous oxide of soluble guanlyate cyclase probed by resonance Raman spectroscopy. Biochemistry 1997, 36:10155-10160. This insightful study probes the effects of substrate (GTP) and product (cGMP) in the presence of Mg2+ on the heme and on its bonding and nonbonding interactions with the heme pocket of soluble guanylate cyclase (sGC). There were no apparent effects of GTP or cGMP on the Fe-histidine bond of unstimulated sGC, the Fe-NO bond of sGC-NO, or the Fe-CO bond of sGC-CO, as judged by insensitivity of their resonance Raman shifts to GTP or cGMP. Only the vNo band was perturbed, suggesting that cGMP is bound in the distal heme pocket or near enough to influence the coordinated NO.

l

10.

Fan B, Gupta G, Danzrnger RS, Friedman JM, Rousseau DL: Resonance Raman characterization of soluble guanylate cyclase expressed from Baculovirus. Biochemistry 1998, 37:1178-l 184.

11.

Yu AE, Hu S, Spiro TG, Burstyn JN: Resonance Raman spectroscopy of soluble guanylyl cyclase reveals displacement distal and proximal heme ligands by NO. I Am Chem Sot 1994, 116:4117-4118.

of

12. .

The major advances in the knowledge of heme-based sensor proteins described herein have followed from synergistic application of multidisciplinary thinking and experimental methodologies. This approach will undoubtedly continue to advance the field through physiological, structural and spectroscopic studies.

Mayer B, Pfeiffer S, Schrammel A, Koesling D, Schmidt K, Brunner F: A new pathway of nitric oxide/cyclic GMP signaling involving Snitroglutathione. J Biol Chem 1998, 273:3264-3270. This paper reports a series of in vitro experiments that provide convincing evidence for the viability of glutathione as an intercellular chaperone for NO. NO synthase-derived NO is shown to activate soluble guanylate cyclase and cause accumulation of cGMP in the presence of superoxide dismutase or glutathione. In the former case, O,- is scavenged and allows NO to diffuse to its target before it reacts with O,- to yield ONOO-. In the latter case, NO and O,- react with glutathione to produce S-nitrosoglutathione, which has a sufficient lifetime to allow the NO unit to reach its target. Release of NO requires Cu+ or an undiscovered enzyme.

Acknowledgements

13.

The author thanks Gudrun Lukat-Rodgers for helpful discussions during the preparation of this manuscript. The author’s work on FixL is supported by the United States Department of Agriculture (awards 96-35305-3628 and 97-35305-5158) and the Hermann Frasch Foundation (award 446-HF97).

Hule RF, Padmaja S: The reaction of NO with Radical Res Comm 1993, l&l 95-199.

14.

Kissner R, Nauser T, Bugnon P, Lye PG, Koppenol WH: Formation and properties of peroxynitrite as studied by laser flash photolysis, high pressure stopped flow and pulse radiolysis. Chem Res Toxicoll997, 11 :1285-i 292.

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Friebe A, Wedel B, Foerster J, Schutz G, Koesling conserved cysteines of soluble guanylyl cyclase. 1997,36:1194-l 198.

J, Malkewitz J, Bohme E, in the PI subunit yields guanylyl cyclase. Proc

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Yunde Z, Marietta MA: Localization of the in soluble guanylate cyclase. Biochemistry 36:15959-15964.

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heme binding 1997,

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of heterodimeric 1995,

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Schrammel A, Pferffer S, Schmidt K, Koesling D, Mayer 8: Activation of soluble guanaylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione. MO/ Pharmacoll998,54:207-212. .~.. This is a compamon study to that reported rn t12’1. It shows that glutathrone reacts with NO and O,derived from the nitrovasodilator prodrug, mosidomine, in vitro to yield S-nitrosoglutathione. NO was liberated by exogenous Cu+ whereupon it activated soluble guanylate cyclase (sGC) and accumulation of cGMP. This chemistry was not inhibited by CO,, suggesting that ONOOis not serving to shuttle the NO unit between the drug and sGC. This adds compelling evidence to the notion that glutathione can serve as a viable intracellular NO shuttle. 17.

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Stamler JS, Jia L, Eu JP, McMahon TJ, Demchenko IT, Bonaventura Gemert K, Piantadosi CA: Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 1997, 276:2034-2037.

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Chan NL, Rogers PH, Amone A: Crystal structure of the S-nitroso form of liganded human hemoglobin. Biochemistry 1996, 37:16459-16464. A high-resolution X-ray crystal structure of S-nltroso hemoglobin A (HbA)-NO is presented. This structure leaves little doubt that S-nitroso HbA forms and can have a substantial lifetime. The carboxy-terminal dipeptide of the chains in S-nitroso HbA-NO is not evident in the structure because it is extruded from its normal R-state position into the solvent by the S-nitroso group. This disruption results in an angle between the alPI and a& dimers that is only 0.6” different from that of the R2 quaternarv structure. Hence, Snitrosation is expected to diminish the affinity’of HbA ior 0, 21.

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Kharitonov VG, Sharma VS, Magde D, Koesling D: Kinetics of nitric oxide dissociation from five- and six-coordinate nitrosyl hemes and heme proteins, including soluble guanylate cyclase. Biochemisfrv 1997. 36:6614-6616. On the basis of kihetic measurements of NO dissociation, the in viva dissociation reaction is estimated to have a half-life of not less than 2 minutes. Faster dissociation is suggested to require an as-yet undiscovered effector molecule.

23. Dierks EA, Burstyn JN: The deactivation of soluble guanylyl cyclase . by redox-active agents. Arch Biochem Siophys 1996,351 :l -7. This studv exolores the oossibilitv of oxidative inactivation of soluble auanvlyl-cyclas;! (s&)-NO. Several dxldants are tested and shown to deactivaie sGC-NO, but none diminished the basal activity of the enzyme. The rate of deactivation tracked the rate of reaction with oxidant and the rate of appearance of ferric heme, but did not correlate with redox potential. This suggests that formation of ferric heme deactivates the enzyme. 24.

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Burstyn JN, Yu AE, Dierks EA, Hawkins BK, Dawson JH: Studies the heme coordination and ligand binding properties of soluble guanylate cyclase (sGC): characterization of Fe(lI)sGC and Fe(lI)sGC(CO) by electronic absorption and magnetic circular dichroism spectroscopies and failure of CO to activate the enzyme. Biochemistry 1995, 34:5696-5903.

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Serfass L, Burstyn JN: Effect of heme oxygenase inhibitors on soluble guanylyl cyclase activity. Arch Biochem Biophys 1996, 359:6-l 6. This study shows that the heme oxygenase (HO) inhibitor tin protoporDhvrin IX stimulates soluble auanvlvl cvclase (sGC) at concentrations below what is necessary to bind in the prosthetic heme pocket, whereas zinc protoporphyrin IX (also an HO inhibitor) inhibits sGC stimulation. The results of this study suggest that the interpretation of physiological experiments employing such inhibitors should be tempered by the realization that enzyme-inhibiting drugs are not necessarily specific. I

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Wu CC, Ko FN, Kuo SC, Lee FV, Teng CM: platelet aggregation through NO-independent soluble guanylate cyclase. Br J Pharmacol

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37. Friebe A, Koesling D: Mechanism of YC-l-induced activation of .. soluble guanylate cyclase. MO/ Pharm 1996, 53:123-l 27. The combined effects of CO and YC-1, a fury1 benzylindazole drug, on soluble guanylyl cyclase (sGC) activation are reported. Based on UV-visible spectra, it is concluded that YC-I binds to the enzyme, but not to the heme. Activitv assavs were used to orobe the nature of the sGC-YC-1 interaction. Data suggeit that YC-1 binbs to sGC in the resting and NO forms, that YC-1 decreases the dissociation rates of NO and CO. The authors suggest that the diminished ligand dissociation rates may be responsible for the increased activation of sGC by YC-1. 36. 0

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Stone JR, Marietta MA: Synergistic activation of soluble guanylate cyclase by YC-I and carbon monoxide: implications for the role of cleavage of the iron-histidine bond during activation by nitric oxide. Chem Viol 1996, 5:255-261. The effects of YC-1 on the binding of NO and CO to soluble guanylyl cyclase (sGC) are probed by direct-rate measurements. The results clarify and/or confirm several important points: YC-1 + CO provides the same level of sGC stimulation as NO: YC-1 has no measurable effect on the rate constants for binding (k,,) or release (&) of CO; the Fe-imidazole bond remains intact in the presence of CO and YC-1. The authors also make an important point that maximal sGC stimulation by the combination of CO and YC-1 requires a partial pressure of CO beyond that which is likely to be provided endogenously by heme oxygenase. l

role for 1995,

29. .

Yet S-F, Pellacani A, Patterson C, Tan L, Folta SC, Foster L, Lee W-S, Hsieh D-M, Perrella MA: Induction of heme oxygenase-I expression in vascular smooth muscle cells. J Biol Chem 1997, 272:4295-4301. This in viva/in vitro rat study provides evidence that inducible heme oxygenase-1 activity in rats treated with endotoxins is increased 6.9.fold. The CO produced in the subsequent heme-oxygenase-catalyzed degradation of heme is shown to exacerbate reduction in vascular tone caused by the NO from inducible NO synthase. This effect is blocked upon treatment with the heme oxygenase inhibitor, Zn protoporphyrin IX. Zakhary R, Poss KD, Jaffry SR, Ferris CD, Tonegawa S, Snyder SH: Targeted gene deletion of heme oxygenase-2 reveals neural role for carbon monoxide. Proc Nat/ Acad Sci USA 1997,94:1464614653. Heme oxygenase 2 (HO-2) and neuronal nitric oxide synthase (nNOS) knockout mice were used to study the coupling of HO-P-derived CO and cGMP builduo in intestinal smooth muscle tissues. The conclusion of couPI&g was based on intestinal smooth muscle relaxation due to cGMP buildup in response to HO-2 activity. Correlation between cGMP buildup and HO-2 activity was established by treating wild type (wt), and knockout (nNOSM* and HO-2u*I mice with drugs to inhibit nNOS (N%itro-L-arainine, L-NNA) and/or Hd-2 (tin protopoyphyrin IX, SnPPIX). Relaxation was observed in nNOSM* mice unless SnPPlX was administered to inhibit CO production by HO-2. Relaxation was observed in HO-2*/A mice unless LNNA was administered to block NO production by nNOS. Diminished relaxation was observed in wl mice upon treatment with either drug and was nearly abolished upon treatment with both drugs. In all cases where relaxation was inhibited, it could be restored to maximal values by treatment with

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for binding of imidazole to the heme depend on the distal sidechain volume. A plot of log /6” versus volume of the Mb E7 sidechain is linear, indicating that the activation barrier height is determined by the steric bulk of the distal E7 sidechain. Based on the rate of imidazole association with ferric RmFixLH, the authors conclude that the distal pocket is unhindered. 56.

Gilles-Gonzalez MA, Gonzalez oxygen sensor FixL depends Biochemistry 1995, 34:232-236.

57.

Nakamura H, Saito K, Ito E, Tamura K, Terumasa T, Nishigaki K, Shiro Y, lizuka T: Identification of the hydrophobic amino acid residues required for heme assembly in the Rhizobial oxygen sensor protein FixL. Biochem Biophys Res Commun 1998. 2471427-431.

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Gonzalez G, Gilles-Gonzalez MA, Rybak-Akimova EV, Buchalova M, Busch DH: Mechanism of auotxidation of the oxygen sensor FixL and Ap/ysia myoglobin: implications for oxygen-binding heme proteins. Biochemistry 1998,37:10188-10194.

46. ..

Gong W, Hao B, Mansy SS, Gonzalez G, Gilles-Gonzalez MA, Chan MK: Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. froc Nat/ Acad SC; USA 1996, 95:15177-l 5162. Single crystal X-ray structures of ferric BjFixLH (i.e. the heme domain of B. japonicum) and ferric B. japonicum FixL-CN reveal that the heme domain exhibits the now ubiquitous PAS (i.e. domains containing two direct sequence repeats [S, and So boxes] of -50 residues each) fold. Differences between the two structures suggest that signal transmission involves largescale motion of the FG loop, especially at Asp21 2. Whether this ligationinduced conformational change is coupled to its own kinase domain or that of a partner in a dimer cannot be addressed by these structures. 47.

Monson EK, Ditta GS, Helinski of Rhizobium meliloti. J B/o/

4%.

Tamura K, Nakamura H, Tanaka Y, Oue S, Tsukamoto K, Nomura M, Tsuchiya T, Adachi S, Takahashi S, lizuka T, Shiro Y: Nature of endogenous ligand binding to heme iron in oxygen sensor Fixl. J Am Chem Sot 1996,118:9434-9435.

49.

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DR: The oxygen sensor Chem 1995, 270:5243-5250.

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Winkler WC, Gonzalez G, Wrttenberg JB, Hile R, Dakappagari Jacob A, Gonzalez LA, Gilles-Gonzalez M: Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide fluoride to the Rhizobial heme-based oxygen sensor FixL. l3iol1996,3:841-850. Rodgers KR, Lukat-Rodgers GS, Barron JA: Structural ligand discrimination and response initiation in the oxygen sensor FixL. Biochemistry 1996, 36:9539-9548.

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51. .

Lukat-Rodgers GS, Rodgers KR: Spin-state equilibria and axial ligand bonding in FixL hydroxide: a resonance Raman study. J Biol lnorg Chem 1998,3:274-281. Adducts between ferric FixL and hydroxide ion exist as thermal equrltbria between high-spin and low-spin six-coordinate species at physiologically relevant temperatures. The presence of the kinase does not significantly perturb this equilibrium to a degree detectable by the vFeoH resonance Raman intensities. 52. .

Lukat-Rodgers GS, Rexine JL, Rodgers KR: Heme speciation in alkaline ferric FixL and possible tyrosine involvement in the signal transduction pathway for regulation of nitrogen fixation. Biochemisfry 1998, 37:1354313552. UV-visible and resonance Raman spectra are reported as a function of pH for RmFixLN (the heme domain form Rhizobinm melilofi FixL) and f?mFixL* (the heme kinase from R. meliloti FixL). The data suggest that heme-kinase interactions do not perturb the heme pocket to such an extent that it causes a detectable change in the high-spin : low-spin (LS) ratio. However, slight differences in pK, values for RmFixLN and RmFixL* suggest that the kinase has some effect on acrd/base properties of the heme and/or one tyrosine residue, whose pK, is indistinguishable from the heme by spectrophotometric titration. While the previously reported kinase inactivation curve apparently tracks formation of heme hydroxide in RmFixL [56], loss of kinase activity is not fully accounted for by the fraction of LS hydroxy heme formed. These data suggest the neutral tyrosine residue that is deprotonated concurrently with formation of the heme hydroxide is required for stabilizing protein conformation necessary in signal transmission. 53.

Bertolucci C, Ming L-J, Gonzalez G, Gilles-Gonzalez MA: Assignment of the hyperfine-shiid IH-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti. Chem Viol 1996, 3561-566.

54. .

Lukat-Rodgers GS, Rodgers K: Characterization of ferrous FixLnitric oxide adducts by resonance raman spectroscopy. Biochemistry 1997, 36:4178-4 187. This resonance Raman study reveals that RmFixLN (the heme domain of R. me/i/of&NO and RmFixL* (the heme kinase of R. me/i/ot&NO exist as equilibria between five-coordinate and six-coordinate low-spin complexes. This distribution between coordination states is consistent with the intermediate strength of the proximal Fe-imidazole (ImH) bond, as indicated by the vFe-rmH frequency of 211 cm-t for deoxyRmFixL, which is the intermediate between myoglobin and soluble guanylyl cyclase (sGC). Formation of a sGC-type nitrosyl heme in FixL suggests the possibility of an NO-induced signal akin to that of sGC. Although this is possible in light of the reported results, there is no physiological evidence for such a signaling pathway. Mansy SS, Olson JS, Gonzalez G, Gilles-Gonzalez MA: lmidazole is a sensitive probe of steric hindrance in the distal pockets of oxygen-binding heme proteins. Biochemistry 1998, 37:12452-l 2457 Kinetics of imidazole binding to various distal pocket myoglobin (Mb) mutants and RmFixLH (heme domain of R. melilofi FixL) are reported. Rate constants

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Zhulin IB, Taylor BL, Dixon R: PAS domain S-boxes in archaea, bacteria and sensors for oxygen and redox. Trends Biochem Sci 1997, 22:33 l-333. This study of sequence homology has revealed 54 PAS domains (i.e. domains containing two direct sequence repeats [S, and Ssl of -50 residues) from all kingdoms. These domains contain both the S, and S2 boxes separated by helical structures. Nearly all the identified structures are sensory domains, suggestrng a broad versatility of this fold m signal transduction mechanisms. 61. .

Pellequer J-L, Wager-Smith KA, Kay SA, Getzoff ED: Photoactive yellow protein: a structural prototype for the three-dimensional fold of the PAS domain superfamily. Proc /Vat/ Acad Sci USA 1998,95:5884-5890. Although photoactive yellow protein (PYP) has a truncated PAS sequence, containing only the S, box and the central helix, it resembles FixL in that its cofactor is located on the central helix near the carboxyl boundary of the S, box. Based upon locations of the heme in FixL and the 4-hydroxycinnamyl chromophore in PYP, the positions of their cofactors in the cavity of the fold are separated by about nine residues. The 4-hydroxycinnamyl cofactor is located at the end of the internal cavity near the amino terminus of the central helix, whereas the heme in the heme domain of 6. japonium FixL is closer to the center of the cavity and over the center of the helix. 62. .

Genick UK, Borgstahl EO, Ng K, Ren Z, Pradervand C, Burke PM, Srajer V, Teng T-Y, Schildkamp W, McKree DE et a/.: Structure of a protein photocycle intermediate by millisecond time-resoved crystallography. Science 1997, 275:1471-l 475. This elegant time-resolved crystattographic study reveals the protein conformational changes coupled to light-induced conversion of the trans 4-hydroxycinnamyl chromophore to its cis conformation. The versatility of the PAS fold is evident by the fact that the light-induced photoactive yellow protein conformational switch is localized at the opposite end of the PAS cavity from that observed for the heme domain from 8. japonicum FixL. 63.

Kerby, RL, Hong SS, Ensign SA, Coppoc Ll, Ludden PW, Roberts GP: Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system. J Bacterial 1992, 174:5284-5294.

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Shetver D, Kerby RL, He Y, Roberts GP: CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein. Proc Nat/ Acad Sci USA 1997, 94:11216-l 1220. CooA from its native organism, R. rubrum, is shown to exist as a dimer in both the CO-bound and free states. The protein was isolated with 1.6 hemes per dimeric unit. It is suggested that some heme may be lost during purification and that the holoprotein contains two hemes per dimer. CooA was shown by DNase I footprinting to exhibit sequence-specific DNA binding only in the presence of CO. The CO bound protein exhibits UV-visible signatures consistent with a sixcoordinate low-spin ferrous heme containing CO and imidazole ligands.

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Aono S, Ohkubo K, Matsuo T, Nakajima H: Redox-controlled ligand exchange of the hemem in the CO-sensing transcriptional activator CooA. J Biol Chem 1998, 273:25757-25764. Site-directed mutagenesis of Cys75 and His77 were shown to affect the electronic spectrum of the heme in CooA. Several conclusions are drawn based on electron spin resonance, UV-visible and transcriptional activation data; Cys75 is an axial ligand in the ferric form of CooA, but not in the CObound ferrous heme; Cys75 is either protonated or unbound in the CO-free ferrous protein; and His77 is the proximal ligand in the CO-bound ferrous heme and probably in the ferrous heme, but is not coordinated to the ferric heme. 72. .

Uchida T, lshikawa H, Takahashi S, lshimori K, Morishima I, Ohkubo K, Nakajima H, Aono S: Heme environmental structure of CooA is modulated by the target DNA binding. J Biol Chem 1998, 273:19968-19992. Resonance Raman and transient absorption results show that sequencespecific DNA-binding elicits a new conformation of the heme or its environment. This conclusion is based on the appearance of new Raman bands in the presence of target DNA and changes in the largest rate constant and relative amplitudes of three kinetic phases observed during CO recombination subsequent to photolysis. The authors were unable to detect the vFe-,m,dazole band of photo-dissociated CooA in the heme resonance Raman spectrum.

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Huang LE, Gu J, Schau M, Bunn HF: Regulatin of hypoxia-inducible factor 1 is mediated by an Os-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Nat/ Acad Sci USA 1996, 95:7987-7992. An oxygen-dependent degradation (ODD) domain within the hypoxiainducible factor 1 (HIF-I) subunit is identified and characterized. This domain controls oxidatively sensitized degradation by the ubiquitin proteasome pathway. The ODD domain comprises 200 residues located in the central region of HIF-1. Deletion of the entire region confers oxygen-independent HIF-1 stability. Thts protein is capable of homodimerization, DNA binding and transactivation in the absence of hypoxic signaling. 81. .

Goldberg MA, Dunning SP, Bunn HF: Regulattn of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1998, 242:1412-l 415. This seminal paper is the first report of compelling evidence that the 0, sensor in the mammalian hypoxic response is a heme protein. The proposal that a heme protein is involved was based on changes in erythropoietin production by the Hep3B cell line in response to treatment with Co*+, Ni*+ and CO. The divalent metal ions were shown to stimulate erythropoietin (Epo) production through a common pathway consistent with transmetallation of a heme protein. Conversely, CO inhibited Epo production by locking the putative sensor in an oxy-like state. 82.

Ho VT, Bunn HF: Effects of transition metals on the expression of the erythropoietin gene: further evidence that the oxygen sensor is a heme protein. Biochem Biophys Res Commun 1996, 223:175-l 60.

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