Dinuclear non-heme iron centers: structure and function

Dinuclear non-heme iron centers: structure and function

Dinuclear non-heme . iron centers: structure and function JoAnne Stubbe Massachusetts Institute of Technology, Cambridge, Massachusetts, USA ...

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Dinuclear

non-heme . iron centers: structure

and function

JoAnne Stubbe Massachusetts

Institute

of Technology,

Cambridge,

Massachusetts,

USA

Proteins containing dinuclear non-heme iron centers have been characterized that catalyse reversible binding of O,, hydroxylation of alkanes, and oxidation of tyrosine to a tyrosyl radical. Physical biochemical methods have revealed novel aspects of both the structure and function of these proteins. Characterization of these centers has been greatly assisted by structural and functional bioinorganic models.

Current

Opinion

in Structural

1991, 1:788-795

Introduction

Hemerythrin

Dinuclear non-heme iron centers have a chemical versatility greater than the ubiquitous and well characterized heme iron systems [ 1,2]. However, their structural elucidation and the characterization of their chemical reactivities have lagged far behind their ostentatious cousins. The diversity of dinuclear non-heme iron-dependent systems is indicated in Table 1. Understanding how nature uses the small repertoire of amino acid ligands and the unique three-dimensional architecture of each protein to define these diverse catalytic capabilities remains a mystery and a major objective of scientists investigating heme as well as non-heme iron chemistry. This review will focus on recent advances in understanding the structure, mechanism and regulatory aspects of a subgroup of these dinuclear iron-centered proteins, specifically on those proteins for which their chemistry requires the interaction of their diferrous forms with molecular Oz.

The best characterized and the prototype for these dinuclear iron-centered protein systems is hemerythrin (Hr), a reversible 0, binder and transport protein found in the blood of marine invertebrates [ 1,2]. This nonheme iron core serves a function similar to that of the heme of hemoglobin in mammalian systems. Myohemerythrin, a monomeric protein from Thermisfe m roides, and hemerythrin, an octamer from 7lwrmiste dyscritum, have been characterized by X-ray crystallographic methods.

The basis for understanding structure and function of metals in any protein is directly related to the level of sophistication of the available bioinorganic model systems. In 1983, seminal papers by Lippard and colleagues [3] and Wiekhardt et al. [4] produced the first structural models for the dinuclear iron cores. These models have proven invaluable to understanding the structural variations that accompany the interconversion of various forms of hemerythrin (Fig. 1) and have given birth to a subdivision of bioinorganic chemistry which has grown exponentially during the past five years. This field has recently evolved to provide models of functional [5**,6,7*,8**,9,10,11*] as well as structural aspects of the dinuclear iron centers.

Ef’R-electron

paramagnetic

Hr-hemerythrin;

Interconversion

MM-methane RNR-ribonucleotide

of deoxy-

and oxyhemerythrin

Recently, Holmes et al. [ 121 reported the structures of deoxy- and oxyHr, the physiologically relevant forms, at 2.OA resolution. There appear to be no major structural differences between the protein portions of deoxyHr or oxyHr and the protein portions of metHr and metazidoHr reported earlier (Fig. 1). Any minor differences appear to reside in the active site. The structural information on the fully oxidized and reduced forms of the protein is consistent with the proposal that O2 binds to the five coordinate iron in deoxyHr. Subsequently, electron transfer from the irons and proton transfer from the OH bridge of the diferrous core occur to produce a hydroperoxyHr (oxyHr) in which the hydrogen on the peroxy moiety is proposed to be hydrogen bonded to the 0x0 bridge. Facile proton transfer between these two moieties may play a crucial role in the reversibility of O2 binding. The mechanism of the conversion of deoxy- to oxyHr is presently unknown. Recent studies using circular dichroism and low-temperature magnetic circular dichroism [ 131 have provided a reasonable explanation

Abbreviations resonance; Et-ethyl; EXAFS-extended

HzHBAR-1,2-bis(2-hydroxylbenzamido)benzene;

788

Biology

X-ray absorption fine structure; HPT~N,N,N’,N’-tetrakis(2-benzimidazole)methyl-2-hydroxy-1,3-diaminopropane; mono-oxygenase; NDP-nucleoside diphosphate; OBz-benzoate; reductase; TPA-tris (2-pyridylmethyl)amine.

@ Current

Biology

Ltd ISSN 095!&44OX

Dinuclear

Table

1. Catalytic

comparison

with

diversity heme

of dinuclear

Dinuclear

0,

iron

binding

non-heme

Heme

Hemerythrin

Myoglobin, hemoglobin

Monooxygenase

Methane

mono-

Cytochrome

iron centers:

structure

and function

Stubbe

for the structure and chemical reactivity of two non-physiological forms of hemerythrin - the (semimet)@ form produced by one electron oxidation of deoxyHr and the (semimet)&-Ir form produced by one electron reduction of the met aquoHr (Fig. 1). It has been suggested that (semimet)& is rapidly reduced because of a minimal requirement for structural reorganization, whereas the (semimet)&+ is rapidly oxidized as a result of the OHligand assisting deprotonation of the OH-bridge.

in

iron proteins.

Function

Reversible

non-heme

non-heme

P450

oxygenase Peroxidase

Ribonucleotide

Electron

transfer

Phosphatase

Prostaglandin

reductase

synthetase

Ruberythrin

Cytochromes

Purple

acid

H

Ribonucleotide

Ribonucleotide reductases (RNRs) cataiyse the conversion of nucleoside diphosphates (NDPs) to deoxyNDPs and hence play an essential role in DNA biosynthesis [ 14.1. RNR is the second best characterized of the dinuclear iron-core proteins. The enzyme is composed of

I

phosphatase

/O\

0

\ His

reductases

H

H H I

,

His

/OZ<

yF$z,-,,3<
His

His &+-

-L \

DeoxyHr

OxyHr

(!Semimet),Hr \ e-

H+

II

N3-

/ Met

azidoHr

\

Met

\

aquoHr

\

N3-

-N=N=N \ His

I,

, /02j

f-e’+

Fe3+

His

A

His

His

Fig. 1. Interconversion

of various

forms

OH’

of hemetythrin

(Hr).

Adapted

from

12,131.

(Semimet),Hr

789

790

Catalysis

and regulation

two types of subunits, each composed of two identical polypeptides: Br (cr,a> and B2 (p,p>. The Br-subunit binds the NDP substrates and contains the allostericbinding sites that govern the substrate specificity and turnover rate, and the thiols that are essential for the reduction process. The Bz-subunit contains the dinuclear iron center and a tyrosyl radical cofactor (Y., using the amino acid one-letter code) essential for reduction. Over the past decade, physical biochemical studies have revealed a great deal about the structure of the cofactor. These studies were corroborated and extended by the recent report of the three-dimensional structure of the Bz-subunit using X-ray crystallography [ 15**]. The structure of reduced Y. and the dinuclear iron center is shown in Fig. 2. 8everal aspects of the structure are novel. The iron center is not triply bridged as in the case of hemerythrin, but is doubly bridged with the residue Glu238 being too far away to be directly coordinated to Fe1 (see Fig. 2). Both irons contact Iive ligands from the protein, their sixth ligand apparently being derived from solvent. In addition, Asp84 is bidentate. Most striking, and in contrast with hemerythrin, only a single imidazole is coordinated to each of the iron centers, the other ligands beiig supplied by carboxylates or solvent. These diIferences, which were anticipated by X-ray absorption line structure (EXAFS) and NMR studies, are consistent with the differences between the functions of these proteins [1,2]. In addition, Tyr122, the site of Y., is located 5.3A from Fe,, consistent with the original proposal that ferrous ions and 02 play an important role in the generation of this protein radical [I6]. The Y. cofactor is also at least 10 A from the nearest surface (i.e. the surface thought to interact with the Br-subunit), making it unlikely that it plays a direct role in NDP reduction. An alternative model has been proposed suggesting that the function of Y. is to generate by long-range electron transfer a protein radical on the Bt-subunit that initiates NDP reduction by hydrogen atom abstraction from the 3’ position of the nucleotide [14*,15-l. Very recent studies have prompted Reichard and coworkers [ 17e*] to suggest that formation and destruction of this essential Y. of Exhericbia coli RNFt might be a no-

u\ / O’

vel method of regulating nucleotide reduction. They have shown in E. coli that the reduced Ravin generated by a flavln reductase is capable of reducing Y. to Y, yielding an inactive form of the enzyme. This same flavin reductase in conjunction with a second poorly resolved protein fraction, for which Fe2+ can substitute, is capable of regenerating the active cofactor. The reductase appears to be able to mediate reduction of the diferric form of the protein to the diferrous form which, in the presence of 02, is capable of rapidly regenerating the dinuclear iron center and Y.. These results are consistent with the observation from the early studies of Atkin et al. [ 161 that apoB2 in the presence of Fe2 + and O2 is capable of generating the active cofactor. The importance of this ilavin reductase in the formation and destruction of the essential Y. is currently being investigated by genetic analysis (P Reichard, personal communication). The demonstration of an absolute requirement for flavin reductase would support the proposed novel regulatory mechanism for controlling nucleotide reduction. The observation that the diferrous form of B2 in the presence of O2 can catalyse Y. formation suggests that this binuclear iron-centered protein may possess catalytic capabilities similar to those of the well characterized hemedependent peroxidases. On the basis of the mechanistic insight provided by studies of heme peroxidases and heme model systems, a number of investigators have suggested the following mechanism for conversion of the diferrous form of the protein to the active cofactor (Fig. 3) [ 18-201. The first step would be analogous to the well characterized conversion of deoxyHr to oxyHr. Once the non-heme iron hydroperoxide is produced, it is suggested that heterolytic cleavage of the oxygenoxygen bond results in formation of a putative Fe”=O.F@ (Fen’ = O.FeN) bond which would be capable, in analogy with compound I or II of horseradish peroxidase, of oxidizing Y122 to Y.. Subsequently, one electron reduction would generate the active cofactor. Recent studies have delined the need for an additional electron in this process in both the mammalian and E. colt’ reconstitutions of the M2- and B2-subunit, respectively [ 18,21**].

Tyrl22

0

Clu23t-I

His241

Glu204

Fig. 2. Active ribonucleotide [15-l.

cofactor of Escherichia reductase. Adapted

co/i from

Dinuclear

Reduced B,-subunit

Fig. 3. Proposed reaction for formation subunit is that subunit of ribonucleotide for reduction. Published with permission

non-heme

of the active cofactor of ribonucleotide reductase which contains the dinuclear [191.

The second intermediate, X, observed by Bollinger et al. [21**] in the assembly of the cofactor of B2 appears to be formed after the formation of the 568~1 species and has a L,.,,, at 360~1. This intermediate also possesses a novel sharp EPR singlet signal at g = 2.00 which is broadened when s7Fe2+ is used in place of 56Fe2+ in the reconstitution experiment. Results from additional characterization by rapid-freeze quench Mossbauer spectroscopy suggest that X contains two high-spin ferric centers coupled to a radical with a spin of 0.5 [22**]. Characterization of X suggests, in contrast to expectations based

structure

Ferry1 intermediate

Peroxide intermediate

Bollinger et al. [21**,22] studying the reconstitution of apoBz with Fez+ and O2 have provided the first mechanistic insight into the assembly of any complex metallo cofactor. Using stopped-flow visible spectroscopy, rapidfreeze quench electron paramagnetic resonance CEPR) spectroscopy and rapid-freeze quench Mossbauer spectroscopy, their studies have revealed the existence of two kinetically competent intermediates in the formation of the diferric core and Y.. Both intermediates appear to be capable of oxidizing Y122 to Y.. Rapid mixing of apoB2 and O2 with Fe?+ results in a transient absorbance at 568~1 (E = 15OOM-tcm-*). This intermediate has not yet been characterized using additional physical biochemical methods; its spectrum, however, is remarkably similar to that of a model compound recently prepared by Que and coworkers [ 5.01 by reacting the diferrous compound Fe2(N-Et-HPTB)OBz(BF4)2 [Et, ethyl; HPTB, N,N,N’,N’-tetrakis(2-benzimidazole)methyl2-hydroxy-13diaminopropanel in CHCl, with O2 at -WC. The product of this reaction had a h,, = 588 nm (E = 1500M-1cm-t). Resonance Raman spectroscopy and Mossbauer spectroscopy are consistent with the assignment of the structure to a j.t 1,2- or u l,lperoxo-bridged species (Fig. 4). Additional putative f.tperoxo-bridged species have recently been prepared [lo]. Further characterization of these species by X-ray crystallography is eagerly awaited.

iron centers:

reductase from apo-B2-subunit, iron center and tyrosyl radical

and function

Stubbe

Active B+ubunit

FeZ+ and Oz. The B2cofactor CTyr) essential

on heme model chemistry (see above) that high-valent iron 0x0 species are not required to oxidize Y to Y.. This provocative result for the reductase system suggests that heme iron chemistry may not serve as a good prototype for non-heme iron chemistry.

Methane

mono-oxygenase

Methane mono-oxygenase (MMO) also possesses a dinuclear iron core and catalyses a most amazing and difficult chemical transformation: the conversion of CH4 to CH30H [1,2,23**,24]. A precedent for this reaction resides with the well characterized heme-dependent cytochrome P450 mono-oxygenases. MM0 has been studied extensively from two sources: Metby1rococcu.scapuhtus and Me&&sinus tricboqmrium. In both cases, the protein requires three components: A (M, -250000), and C (M, - 42 000). The A compoB (M, -17000) nent of the enzyme, a dimer of three subunits, a2p2y2, possesses the mono-oxygenase activity and two dinuclear non-heme iron cores. The C component contains a 2Fe2S center in addition to a &in providing the required reducing equivalents for this process. Component B is required for efficient electron transfer between the A and C components. Fox et aA [23**] using kinetic, spectroscopic and chemical evidence have advanced a model for the structural organization of MM0 in which the three components are tightly associated. The proposed affinity for the MM0 components greatly exceeds that observed in comparable cytochrome P450 systems. A variety of physical biochemical methods, including EXAFS, EPR and Mossbauer spectroscopy, suggest that MM0 contains a bridged dinuclear iron core, with the nature of the bridge at present remaining a mystery [2]. As in the case of hemetythrin, three different redox states of the protein have so far been characterized. The

791

792

Catalysis

and regulation

fully reduced high-spin diferrous state exhibits an integer spin EPR signal, g = 16, similar to those observed with hemerythrin and RNR [ 251. A mixed valency state exhibits an EPR signal, gavg = 1.83, reminiscent of the mixed valency states observed with hemetythrin. The fully oxidized, diferric-natural state, is EPR silent and the di-iron core appears to be weakly antiferromagneticafly coupled. In contrast to both hemerythrin and RNR, the visible spectrum of MM0 is featureless at > 300 nm. These latter two observations in conjunction with Mossbauer and EXAFS [26*] studies suggest that the native diferric state of MM0 does not contain a t.t 0x0 bridge. The coordination environment of the iron core is unknown, although recent sequence analysis of RNR and MM0 has fuelled speculation that the ligands in MM0 may be similar to those in RNR 1271.

.

R I \ IIIIII,,.

/O\

/ Fen .,,,11111

Fe’

4

‘o

o’

\

Y

Recent studies of Lipscomb and coworkers [28] have indicated that, as with hemerythrin and RNR, the diferrous form of the protein is essential for hydroxylase activity, and that component A in the absence of components B and C is capable of converting propylene to propylene oxide. Recent substrate-specificity studies [24,29,30*] have also led to speculation that the mechanism of this non-heme iron mono-oxygenase may be similar to that postulated for cytochrome P450. A typical cycle is shown in Fig. 5. The postulated mechanism is also very similar to that proposed for the formation of the Y. catalysed by RNR (Fig. 3). The major difference is that the oxidation of methane is a two-electron process, whereas oxidation of tyrosine is a one-electron process. Nothing, however, is actually known about this catalytic cycle, and surprises may be revealed as they have been for RNR [ 21**,22**].

fig.

4. Reaction of FezfN-Et-HPTB)OBz(BF& with Oz in CH,CI, at form a u1,2- or ul,l-peroxo-bridged species. Et, ethyl; HPTB, N,N,N’,N’-tetra-kis(2-benzimidazole)methyl-2-hydroxy-1,3diaminopropane; OBz, benzoate; R, part of the HPTB ligand. Published with permission [WI.

-60°C to

Fern,

CHsOH

/

NADH,H+ reductase, component

OH

Fern

FeN, Fern,

?H

l OH

CH,

B

-I--

k7

2es chemical reduction

NAD+ reductase, component

B

Fe: PH Fe’

02

FeN, ?H Fe”\\ ‘“;‘34

0

-;d

W

Fig. 5. A catalytic mono-oxygenase. mission [291.

cycle for methane Published with per-

Dinuclear

CH.q

Pd(m

+ HzOz

CF3C(O)20 9o”c

H

CH *

OH

non-heme

iron centers:

structure

and function

Stubbe

+ H o

3

2

““\ PF3 /p-q -O1 f” H--H3

Fig. 6. Postulated mechanism for PdW catalysed formation of CH3OH. Mn+, metal ion. Published with permission 17.1.

Very recently, several model reaction systems have been reported which begin to define the structures of feasible intermediates in non-heme iron-catalysed hydroxylation reactions. The best characterized structurally is an intermediate observed during the course of alkane functionalization (hydroxylation of cyclohexene) by the reaction of the (u-oxo)diferric complex, Fe2TPA(C10& [TPA, tris (2-pyridylmethyl>amine] with H202 [8**]. The intermediate has been characterized by visible and Raman spectroscopy as well as by EPR and Mijssbauer spectroscopy. These data suggest that the dinuclear ferric starting material has been converted to a mononuclear intermediate formulated as [(LO+ >Few = 0]3+, a species with features similar to compound I observed with heme peroxidases. Although additional structural characterization is required, these observations support the notion that high-valent iron-ox0 species may play a role in alkane hydroxylation mediated by non-heme iron systems. Recently, Stassinopoulos and Caradonna [6] reported the reaction of a dinuclear iron core, [Fe2+(H2HBAE%>(N-methylimidazole)]2 [H&lBAB, 1,2bis(2-hydroxylbenzamido)benzene] with peracids and iodosobenzene and revealed that the same transient intermediate (tl,, = 30-60 min at - 78”) is kinetically competent to convert cyclohexene to cyclohexenol and cy clohexene epoxide. The nature of this transiently formed species and whether or not it contains an Fe” = 0 moiety remain to be elucidated.

Conclusion Within the past year, major breakthroughs in dinuclear non-heme iron chemistry have occurred on two fronts. The first, and the key to successfully unraveling the biological systems, involves development of functional bioinorganic model systems. A variety of ligands are now available which provide an open coordination site(s) on the diferrous form of the core system allowing chemistry to occur with 02. Recent results suggest that both peroxides and high-valent iron 0x0 species have been generated. The next few years will unravel the possible chemistries available to dinuclear non-heme iron systems, a prerequisite for understanding the mechanistic options available to the proteins containing these centers. Secondly, important advances have been made in understanding the protein systems themselves. With RNRs, two novel intermediates have been observed and, although much more detailed structural investigation of these intermediates is required, novel chemistry has already been uncovered. The recent advances made with MM0 suggest that elucidation of this intriguing reaction mechanism using methods similar to those applied to studying reductase will soon follow.

References Finally, Sen and coworkers [7*] have recently described a Pd(II) catalyst that at low temperature can convert methane to methanol. An electrophilic rather than a radical mechanism has been proposed to accommodate the available experimental information (Fig. 6). This type of mechanism would require the intimate involvement of the putative carboxyiate ligands on the dinuclear iron core and would require hydrolysis of a methyl ester, a reaction which has precedent with the purple acid phosphatases (Table 1).

and recommended

Papers of special interest, published have heen highlighted as: . of interest .. of outstanding interest 1.

the annual

period

of review,

Gl+ AVERILL BA: Proteins Ck~taining Iron Centers: a Bioinorganic PerRev l!K’O, 90:1447-1467.

VINCENT JB, OLMER-LU.lEY

Oxo-bridged spective

2.

within

reading

Dinuclear Cbem

QUE L JR, PRUE AE: Diiuclear Iron in Biology. In Progress in Inorganic

and Manganese-oxo them-:

Sites

Bioinmganic

793

794

Catalysis

and regulation

Gbemhtry, Vcf 38, edited by Lippard SJ [book]. New York: John Wiley and Sons, 1998, pp 105-200.

3.

4.

ARlrlslR0~G W-I, SPOOL 4 PAp~~~nwhnous GC, Fw-wL RB, Lwpm SJ: Assembly and Characterization of an Accurate Model for the Diiron Center in Hemerydwin. / Am cban sot 1984, 10636533667.

NORDUTNDP, SJOBERGBM, EKLUND H: Threedimensioti Structure of the Free Radical Protein of Ribonucleotide Reductase. Nature 1990, 3453593598. The first insight into the dinuclear iron center of RNR. Unfortunates, the 30residue carboxy-terminal tail, which appears to play a key role in binding B2 to Br, is not observable. 15. ..

16.

ATK~NCL THELANDERL, REtcw P, LONG G: Iron and Free Radical in Ribonucleotide Reductase. J Bid C&em 1973, 248~7464-7472.

Ww K, POHL K, GEBERT W: [{(C&ItsN3Fe]a (PO&I-CH,CO~)~]~+, a Diiuclear Iron Complex with a Metazidoheoqerythrin-type Structure. AngeW &em Int Ed En@ 1983, 22:727.

17. ..

MENAGES, BUNNAN BA, Jum-G.wu C, MONCK E, QUE L JR: Models for lron-oxo Proteins: Dioxygen Siding to a Diferrous Complex. J Am @em Sot 1990, 11264236425. One of the tirst model systems for the functional chemistry of diferrous systems interacting with molecular 02.

18.

Oauu El, M,w8 GL, G&WND A THEIANDERL: vosyl Free Radical Formation in the Small Subunit of Mouse Ribonucleotide Reductase. J Biol cbem 1990, 265:1575815761.

Sr~sst8cwoutos A, C~twohw JP: Binuclear Non-heme Iron 0x0 Transfer Analogue Reaction System: Observations and Biological hnplications. J Am them Sot 1990, 112:7071-7073.

19.

Uo LC, HUTTONAC, SENA Low Temperature, PaEad.ium(B)Catalyzed, Solution-Phase Oxidation of Methane to a Methanol Derhatiw. /Am ujem Sot 1991, 113:700-701. A novel model system for the oxidation of methane to methanoL

SAH~ M, SJOBERGBM. BACKES G, ~EHR T, SANDE~~L~EHR J: Activation of the Iron-containing Ba Protein of Ribonudeotide Reductase by Hydrogen Peroxide. Biocbem Bi@ys Res C.lwnm 1990, 167:81w18.

20.

FO~CAVE M, GEW C, A~TA M, JEUNET A High Valent Iron 0x0 Intermediate Might be Involved During Activation of Riinucleotide Reductaw Single Oxygen Atom Donors Generate the Q-rosy1 Radical. Biocbem Biophys Res Comm 1990, 168:65*.

5. ..

6.

7. .

LE~SLNG RA, BREMYANBA MUNCKE, QUE L JR: Models for Nonheme Iron Oxygenase: a High-valent Iron-ox0 Intermediate. J Am C&m Sot 1991, 1133988-3990. A hmctional model for MMO: characterization of the first non-heme [(Ls+>Fetv=O>]3+ intermediate.

Forwtc~vs M, GEREZC, M.wsuv D, REICHARDP: Reduction of the Fe(m)-Tyrosyl Radical Center of Escbedcbla calf Ribonucleotide Reductase by Dithiothreito!. J Bid C&m 1990, 265:1091~1C924. An overview of the protein factors and their apparent function in the regulation of nucleotide reduction in E coli.

8. ..

9.

10.

NORMANRE, HOLE RC, MENAGE S, O’CONNOR CJ, ZHANG JH, Qm L JR: Structures and Roperties of Dibridged (p-oxo)diiron(m) Complexes. Effects of the Fe-O-Fe Angle. Inorg Gem 1990, 294629-4637. BRUYNANBA, Ctnth’ Q, Ju~w-G~act~ C, TRUE AE, O’CONNOR CJ, QUE L JR Models for Diin-oxo Proteins: the Peroxide Adduct of Fe2(HFTB)(OH)(N03~). ICbem 1991, 38:1937-1943.

11. .

Tow WB, Uu S, BENI’SENJG, LIPPARDSJ: Models of the Reduced Forms of Polyiron-oxo Roteins an Asymmetric, Triply Carboxylate Bridged Diiron(B) Complex and its Reaction with Dioxygen. JAm &zm Sot 1991, 113152-164. One of the first model systems for the functional chemistry of dinuclear ferrous systems interactin with 02. 12.

Hothiss MA, STRONG I, TuRlEy S, SIEKERLC, SEM(AMP RE: Structures of Deoxy and Oxyhemerythrin at 2.OA Resolution J Moi Bioll991, 218583593. The sttuctures of the physiologically relevant forms of hemetytbrin are reported and support the data accumulated from a IatBe number of physical biochemical experiments.

13.

14.

MCCORMICKJM, SOU~MONEl: Spectroscopic Studies of the Mixed-Went [Fe(II),Fe(m)] Forms of the Non-heme Iron Rotein HemIron Coordination Difkrences Related to Reactivity. J Am C%em Sot 1990, 112:200~2007.

SIUBBE J: Ribonucleotide Reductases Amazing and Confisiq. J Efoi C5em 1990, 265:532+5332. ;he most recent review covering both structural and mechanistic aspects of all dbonucleotkie reductases.

21. ..

BOUINGERJM, EDMOND~~NDE, HUYNH BH, Fnw J, NORTON J, STUBBEJ: Mechanism of Assembly of the Dinuclear Iron Center-Tyrosyl Radical of Ribonucleotide Reductase. Science 1991, 253 292-298. .. The first characterization of the mechanism ot assembly ot a complex metallo cofactor. The process is characterized kinetically via stoppedBow UV-visible spectroscopy and rapid-freeze quench EPR BOLUNGERJM, STLBBEJ, HUYNH BH, EDMONDSONDE: A Novel Diferric Radical Intermediate Responsible for I)rosyl Radical Formation in the Assembly of the Cofactor of R&onucleotide Reductase. J Am them Sot 1991, 113628%291. Rapid freeze-quench Mossbauer spectroscopy is used to characterize a novel intermediate in the assembly of the iron center of E c&i reductax. 22. ..

Fox BG, Ltu Y, DEGE JE, IIpscohm JD: Complex Formation Between the Protein Components of Methane Monooxygenease from Metby1ostnu.r Mchospodum OB3b. J Bid Gem 1991, 266540-550. The three components of MMO, in contrast to camphor cytochrome 450s. appear to be tightly associated. Insight into the complexing partners is provided. This is the first qualitative picture of the structural interactions of this three-component system.

23.

..

24.

GREENL, DALTONH: Substrate Speciticity of Methane Mono0xygenase.J Biol &em 1989, 264:17698-17703.

25.

HEND~UCHMP, MONCK E, Fox BG, Ltpscohm JD: Integerspin EPR Studies of the Fully Reduced Methane Monooxygenase Hydroxylase Component. J Am C&m Sac 1990, 1125861-5865.

26.

D!zwr JG, BE~T.EN JG, ROSENZWEIG AC, HEDMON B, GREEN J, DUUNGTONS, PA~IJEF~~OU GC, DALMN H, HODGSONKO, L~PARDSJ: X-ray absorption, Mossbatter, and EPR Studies of the Dinuclear Iron Center of Hydroxyiase Component of Methene MonooxyRenase. J Am Chn Sot 1991, in press. A variety of physical biochemical methods have been used to invest@te the diferric form asnd mixed valent fotm of MMO.

Dinuclear 27.

STANIHORPEAC, LEESV, SAIMONDGPC, DALTON H, MURIIJXL JC: The Methane Mono-oxygenease Gene Cluster of Metby kxoccus cu@ukatus (Bath). Gene 1990, 91:27-34.

28

Fox BG, FROIANDWA DEGEJE, LIPSCOMBJlI Methane Monooxygenease from Metbylosinw ticbospohm OB3b. J Biol C5em 1989, 264:10023-10033.

29.

RUZICKAG, HUANG DS, DONNELLY ML, FIIEY PA Methane Mono-oxygenease Catalyzed Oxygenation of l,l-Diiethylcyclopropane. Evidence for Radical and Carbocation Intermediates. Biorbemisty 1990, 291696-1700.

non-heme

iron centers:

structure

and function

Stubbe

FOX BG, BORNEMANJG, WACKET?II’, LrpscoMBJDz Hak&etu Oxidation by the Soluble Methane Mono-oxygcnue from Metbylosinus Trfcbospodum OB3bz Me&a&tic and Environmental Implications. @cbem#y 1990, 296419-6427. The chemistry observed is similar to the well characterized P45Os.MM0 is capable of oxidizing chlorinated akenes including the ground-water contaminant trichloroethylene. 30. .

JA Stubbe, Departments of Chemistry and Biology, Room l&288, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

795