Proc. Nati. Acad. Sci. USAVol. 91, pp. 2592-25%, March 1994Biochemistry

Mutation of His-105 in the 13i subunit yields a nitric oxide-insensitiveform of soluble guanylyl cyclaseBARBARA WEDEL, PETER HUMBERT, CHRISTIAN HARTENECK, JOHN FOERSTER, JORGEN MALKEWITZ,EYCKE BOHME*, GONTER SCHULTZ, AND DORIS KOESLINGtInstitut for Pharmakologie, Freie Universitit Berlin, Thielallee 67-73, 14195 Berlin, Federal Republic of Germany

Communicated by David L. Garbers, December 7, 1993 (receivedfor review August 12, 1993)

ABSTRACT Soluble guanylyl cyclase [GTP pyrophos-phate-lyase (cyclizing); EC 4.6.1.21 is a hemoprotein that existsas a heterodimer; the heme moiety has been proposed to bindnitric oxide, resulting in a drmatic activation of the enzyme.Mutation of six conserved His residues reduced but did notabolish nitric oxide stimulation whereas a change of His-105 toPhe in the^ subunit yielded a heterodimer that retied basalcyclase activity but failed to respond to nitric oxide. Heme wasnot detced as a component of the mutant heterodimer andprotophorphyrin IX failed to stimulate enzyme activity. Theactivt Of the His mutant was almost identical to that of thewild-type enzyme in the presence of KCN, suggesting thatdsruption of heme binding is the principal effect of themutation. Thus, the mutation provides a means to inhibit thenitric oxide-sensitive guanylyl cyclase s pathway.

Guanylyl cyclases [GTP pyrophosphate-lyase (cyclizing);EC 4.6.1.2], which catalyze the conversion ofGTP to cGMP,exist in soluble and membrane-bound forms. Membrane-bound guanylyl cyclases belong to a group ofreceptor-linkedenzymes and are stimulated by various peptide hormones (1).The soluble enzyme contains heme as a prosthetic group andis activated by nitric oxide (NO) and NO-containing com-pounds (2, 3). The finding that the heme-free enzyme exhibitsbasal activity but is not stimulated by NO led to the proposalthat enzyme-bound heme acts as the receptor molecule forNO, transducing the binding signal to activation of thecyclase catalytic domain (4). The heme is apparently notcovalently bound as it can be removed from the heme-containing enzyme and also reinserted into a heme-freeenzyme, thereby restoring the stimulation by NO (5).

Soluble guanylyl cyclase is a heterodimer consisting of ana subunit and a (3 subunit (6). Two a and two P subunits(al,a2,A, and (2) have been identified in rat and bovinetissues (7-12), and expression experiments have revealedthat both an a subunit and a ( subunit are required for theformation of a catalytically active enzyme (8, 13). All sub-units contain homologous C-terminal domains (250 aminoacids), which are also homologous with the membrane-boundguanylyl cyclases and with the eukaryotic adenylyl cyclases(1, 14, 15).The aim of the present study was to identify amino acids

involved in the NO-heme-mediated activation of solubleguanylyl cyclase. As His residues are known to have animportant function in binding of the heme moiety in otherhemioproteins, we constructed several His mutants of the aland Pi subunits by using site-directed mutagenesis. Theircharacterization revealed that a single mutation of His-105 inthe Pi subunit yielded an enzyme still able to catalyze theconversion of GTP to cGMP; however, NO no longer stim-ulated the enzyme.

MATERIALS AND METHODSSite-directed mutagenesis ofHis residues was achieved usingthe polymerase chain reaction (PCR). To minimize mistakesintroduced by the PCR, only fragments flanking the particularHis residue were amplified and subsequently used to replacethe respective unmodified fragment in the wild-type cDNA.In the recombinant PCR technique used for the AH7F,(3H105F, P3H134F, and (3H220F mutations, two overlap-ping PCR products, in which the respective mutation hadbeen already introduced by the primers, were purified onagarose gels, denatured, and reannealed to form a heterodu-plex, which was extended and amplified using the left- and

right-most primers in a second PCR. For the AH346F,alH407F, a1H441F, (3H105K, and (31H105R mutations, thesecond PCR was omitted and a new restriction site, whichhad been introduced by the primers in the first PCR, inaddition to the mutation, was used to ligate the PCR frag-

ments. The resulting products were subcloned using restric-tion sites introduced by the outside primers, and fragmentscut by suitable restriction enzymes were used to substitutefor the respective portions in the original cDNAs. Subse-quently, the entire sequence introduced by the PCR, includ-ing the mutation, was verified by sequence analysis. Themutants were subcloned in the expression vector pCMV.The PCR was carried out in 10 cycles (940C, 60 s; 450C, 120

s; 72C, 120 s), by using 1 jg ofcDNA codingforthe respectivesubunit as template, 80 pmol of each primer, and 2.5 units ofPfu DNA polyMerase (Stratagene) under the conditions sug-gested by the supplier. In the second PCR, 5 cycles wereperformed without the oligonucleotides and another 25 cycleswere performed after addition of the primers.

Expression of the modified subunits in COS cells, determi-nation of guanylyl cyclase activity in the cytosol of thetransfected cells, and immunoblot analysis of the cytosolicproteins were performed as described (13). Antibodies againstC-terminal peptides ofthe .81 and al subunits were used for thedetection of the modified and unmodified subunits.For the expression in the baculovirus system, cDNAs

encoding the a, subunit, the (31 subunit, and the (31H105Fmutant were subcloned in the pVL1393 baculovirus expres-sion vector (Invitrogen) as follows. The cDNA of the ai

subunit ligated inEcoRI site ofpBR322 was transferred to theEcoRI site ofpVL1393. For the Pi subunit, the 2.5-#b gpNAligated at the HindUI and EcoRI sites of pBluescript SK(-)

was digested with Xba I and the fragment was cloned to theXba I site of pVL1393. The cDNA of the P3H105F mutantligated at the HindIII and EcoRI site of pBluescript SK(-)was digested with Nae I and EcoRI and the resulting 2.4-kbfragment was ligated at the Sma I and EcoRI site ofpVL1393.Monolayer cultures of fall armyworm ovary (Sf9) cells werepropagated in TNM-FH medium (Sigma) supplemented with

Abbreviations: NO, nitric oxide; SNP, sodium nitroprusside;GSNO, S-nitrosoglutathione.*Deceased June 11, 1993.tTo whom reprint requests should be addressed.

2592

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 91 (1994) 2593

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CLI 60 K::-L-,-::I F.R...:E FE R L N LK -.IQ --R-:-TM-:-------------- AKHKIKEN:: ---,.KSLEREDFEKIVV-D.'Q.AIAA -------7...........

A.:a2 75 -GXAAVQ R:MM7

DJ 1 MYGFVNIIA,--i---,ELLVIRNYGPE---VWEDIKKEAQLDEEGQFLVRIIYDDSKTY ----------------ZI VAAA S KV --.:L::02

... .. .... 10 ........ 2 0 ... ... .. 3 0 ........ 4 0 ....... . 5 0 ........ 60 .... ..70 .... ... . 80 ... ... .. 9 0.... .................

al 127 :G-VP VZI -1-.::K E S L Gkk_".t E.Z.-"D E Y -.--S T:A,.::L K Q S S H C QEAEX K G RF EDX I.: :CLDKD--PDV YVY.-Y: F:PKR: :TSP:F M......... .....

..C.' IRAM lk' aYF,9GF--DKULE--HIRTSFG: QATLES MCK E L P E G Z..." M L ItY HP H 14 VGa2 163 K FEE X. - V. G.--.:T:...

........ ......... 1 0 5 1 3 4... ...... ........

DJ 60 N, N AGE 1:VQM LVI T D A D K G:Y,, G L 1::-LjfXYs-E R E G L.Q D;..'XMF T'.. Q 9..: :S .D:T I-IRV L Z: LA:T I Y G MRAPSZ.m I R -s02 1 :A -X::V:1K L F F.- F. KH A -̀M.-8 s Y...: A L S Y Q E M N: P S: ---- VEEGAD'G-AML X.X: D R H:G.:LC HA ..::...F

... ....... ........ ............... ......

......... 10 ........ 2 0 ........ 3 0 ........ 40 ....... . 50 ...... .. 60 .... .... 7 0 ... .... . 8 0 ..... . .. 90

a, 214 L I L P:0±-l,:.X--,,:,,.,,:,,A......kX ZSTPSRFHQDCREFVDQPCE:: ...!YSVHIRSA-RPHPPPGKP-:VSSLVIP ..CX T7:PY::HYXL-D R..................

a2 248 F AM L G MIX....A.-A G K L R I S L DV V:EQVANEKLCSDVSNPGNCSC': :TFLIKECE-NTNIMKNLP.QGTSQVPAD-LRISINT-.F:C:RAF-:P:F::H-:LM'F-D P...........

.....122:0........ ..... --RN C.:AP1 145 T::'-''."A'-'-QQIHGTEIDMKVIQ--o- -E:ECDHTQFLIEEKESKEEDFY-EDLDRFEENGTQESRISPYT-- 1: DR

P2 82 'KDFFDTDVAMSILDxo::GSE'DSQADQEALQGTLLRMKERY:LNIPVCPGEKSHSTAVRASVLFGK....... ............. ......... .......

..........

......... 10 ....... . 2 0 .. .... .. 3 0 ........ 40 ... ..... 50 ..... ... 60 ........ 7 0 .... ... . 80 ... .. ... 90al 296 D:MS QHO-Ift.: LMSRRDVQGKPHEDEY.k.-'E."..ii.LT:"'::'-'K"'ISQT.:F.:SG:.I-:MTM::LNMQF:::L.V:R VRRWDNSMKKSSRVM'D--L:x 0 YMV'R

....... ....... ...........

S: L R X-14: y 1: 1:r..: V S: X.VN A TT':E RV L L R.: S T P: V ------- T K P E A S G S E N K D KV F,--V' -QQM'T'HVP..E:.... ...

.... ....

.......

226 D..L..V..V.....T.OC-G-NX--:1-:VRVLP-.OLQ-PGNCS::.)L-::LSV::I,:SLVR.P.'HIDISF..HG..1-:LSH':--I-.::'N.."TVF..:..V.: :RSKEGLLDVEKSECEDELGTEISC: -t."K.-` L PZ:.............. ......

02 222 A-L.RV F -.WME S.0AQV Q X Y V-P-G I L T Q K F A: D E Y ..S I I H-P---'.QV T F N I S S: 1-:C K F: S T RK E MM-------- PKARKSQPML: :...:R: QM1

... .. .... 10 . ....... 2 0 ........ 3 0 ........ 40 .... .. .. 50 ...... .. 60 .. .. .... 7 0 ....... 80 .. ...... 90

1l 377 SSSI-:a2 416 SNS..I.

1o 316 AD"s.I02 303 LRCMI

407 44 1 ...

..- -....-:.-.......... S:::::.::.:::: : : :........... ...... ... -.-..... -. .. ...-. .. .-:..-.--.-. .----. .-'

''':' '' ' ''' -' '" ' '' ' :....:.:...............} ~~~~~~~~~~~~~~~~~~..

.........-.-.....-..-....... ...............

--.-.---... ...-.-.-.-.

FIG. 1. Positions ofthe mutated His residues. Shown is the alignment of the four known subunits ofsoluble guanylyl cyclase. The His residuesthat were mutated are in boldface type; the positions ofthe mutations are given above the respective residues. x indicates occurrence ofadditionalamino acids in the P2 subunit. Shaded residues indicate identical amino acids.

10% (vol/vol) fetal calf serum. Spinner-cultured cells weregrown in TNM-FH medium supplemented with 10%16 fetal calfserum, amphotericin B (2.5 gg/ml), streptomycin (100 pug/ml), penicillin (100 units/ml), and 1% lipid concentrate(GIBCO). Recombinant viruses were generated by cotrans-fection of Sf9 cells with the expression vectors describedabove and with BaculoGold baculovirus DNA (Dianova,Hamburg, F.R.G.) by the Lipofectin method (16). Positiveviral clones were isolated by plaque assay and were identifiedby their ability to direct the expression of the appropriateproteins as revealed by immunoblot analysis. For purificationof recombinant guanylyl cyclase, spinner cultures (1.8-2.5liters) grown to 1.5 x 106 cells per ml were coinfected withthe appropriate recombinant baculoviruses at a multiplicity

AkDa

U VWW .- -.

of infection of 5 for each virus. Cells were collected bycentrifugation 38-44 h after infection, resuspended in 4 vol of50mM triethanolamine hydrochloride, pH 7.5/2mM reducedglutathione/1 mM EDTA/0.2 mM benzamidine/1 puM pep-statin A/0.5 mM phenylmethylsulfonyl fluoride, and lysed bysonication. The homogenate was centrifuged at 200,000 x gfor 20 min at 40C, and purification of wild-type and mutantenzyme from the cytosol was as described (6).

Soluble guanylyl cyclase (100 1.l) purified from bovine lung(4.8 pug) and wild-type (4 pg) and mutant (4.8 Mg) enzymespurified from insect cells were injected onto a column (0.5 x6 cm) of Sephadex G-25 fine (Pharmacia) equilibrated with abuffer containing 25 mM triethanolamine hydrochloride (pH7.8) and 300 mM NaCl (flow rate, 0.2 ml/min). Absorbance

B

_ 94 -A---S 67 * mm am "W

- 43 -

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

FIG. 2. Immunoblots demonstrating the expression of the mutated guanylyl cyclase subunits in the cytosol of transfected COS cells. Appliedon parallel 10o SDS/PAGE gels was 0.1 mg of cytosolic proteins from cells cotransfected with the cDNAs of the a, and A subunits (lanes 1and 2), of the al subunit and the P3H7F mutant (lane 3), of the a, subunit and the P1H1O5F mutant (lane 4), of the a1 subunit and the ,31H134Fmutant (lane 5), of the a, subunit and the p8H220F mutant (lane 6), of the a1 subunit and the I31H346F mutant (lane 7), of the alH407F mutantand the PI subunit (lane 8), and of the alH407F mutant and the Pi subunit (lane 9). In lane 10, cytosolic proteins of untransfected COS cellswere applied. The subunits were detected by antibodies against the (31 subunit (A) and by antibodies against the ca1 subunit (B). Lane 1 showsthe antibody reaction in the presence of the respective antigenic peptide.

Biochemistry: Wedel et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

Table 1. Activity of His mutants of soluble guanylyl cyclase expressed in COS cellsGuanylyl cyclase activity, pmol of cGMP per min per min

Expressed Mg Foldmutant - SNP + SNP - SNP + SNP stimulation

Wild type 111 ± 19 (7) 1912 ± 288 (7) 127 ± 29 (9) 3041 ± 518 (9) 26P1H7F 35 ± 5 (5) 192 ± 24 (7) 17 ± 3 (6) 312 ± 26 (6) 22PH105F 62 ± 14 (7) 60 ± 14 (7) 30 ± 10 (9) 32 ± 11 (9) 1P1H134F 18 ± 2 (5) 47 ± 5 (7) 6 ± 1 (3)* 73 ± 20 (7) 12P3H220F 69 ± 9 (7) 1248 ± 113 (7) 64 ± 14 (9) 2294 ± 219 (9) 3481H346F 60 ± 11 (5) 541 ± 78 (5) 8 ± 6 (5) 5% ± 56 (5) 36alH407F 29 ± 10 (3) 222 ± 55 (3) 10 ± 3 (3) 414 ± 86 (3) 43alH441F 54 ± 14 (3) 1072 ± 189 (3) 40 ± 16 (3) 2115 ± 231 (3) 84COS cells were transfected with the cDNAs of the a, and Pi subunits (wild type) or with the cDNA

of the indicated mutants plus the cDNA of the unmodified corresponding subunit of soluble guanylylcyclase. Three days after treatment of the cells, enzyme activity was determined in cytosolic fractionsof the transfected cells with Mn2+ or Mg2+ as indicated in the absence or presence of 0.1 mM sodiumnitroprusside (SNP). Data are the mean ± SEM (number of experiments is in parentheses). Innontransfected COS cell cytosol, guanylyl cyclase activity was undetectable even in the presence ofSNP. Fold stimulation of cGMP production in the presence of Mg2+ and SNP is indicated.*Below the detection limit in four other experiments.

spectra were recorded in a photodiode array detector with an8-i4 sample cell (Waters 990, Millipore).

RESULTSWithin the primary structure of the four subunits of solubleguanylyl cyclase, several His residues are conserved eitheramong the respective isoforms of the a or (3 subunits oramong all subunits. To elucidate whether these residues playcrucial roles in the stimulation by NO, His-7, 105, 134, 220,and 346 of the (31 subunit ((3H7F, P3H105F, 3H134F,P3H220F, and jH346F, respectively) and His-407 and 441 ofthe ai subunit (aiH407F and a1H441F), all of which arelocated outside the putative catalytic domain, were mutatedto Phe (Fig. 1).The point mutations were introduced in the cDNA clones of

the a, and PI subunits. Subsequently, COS cells were cotrans-fected with the cDNA of the mutants and the cDNA for thecorresponding unmodified subunit. Western blots using anti-bodies directed against C-terminal peptides of the a, and 3subunits were used to confirm that modified and unmodifiedsubunits were expressed in comparable amounts (Fig. 2). Allmutants were catalytically active, although basal activitiesmeasured in the presence of Mg2+ as the cation were de-creased by variable extents (Table 1). With the exception ofthe P3H105F mutant, however, all modified proteins also

Table 2. Stimulation of guanylyl cyclase mutants byvarious activators

Expressed Fold stimulationmutant + SNP + GSNO P-IX

Wildtype 21 21 4PiH7F 11 17 9PjH105F 1 1 1P1H134F 5 6 2P1H220F 66 66 11P1H346F 97 101 13alH407F 30 30 10a1H441F 95 100 11

COS cells were transfected with the cDNAs of the a1 and pisubunits (wild type) or with the cDNA of the indicated mutant plusthe cDNA of the respective unmodified corresponding subunit ofsoluble guanylyl cyclase. Enzyme activity was determined in thepresence of0.1mM SNP, 10 pM GSNO, or 1 PM protoporphyrin IX(P-IX). Data represent fold stimultion as compared to unstimulatedcontrol values and are a representative experiment of three experi-ments, performed in triplicate.

responded to SNP with a marked increase in cGMIP formation(12- to 84-fold stimulation). In contrast, mutation ofHis-105 ofthe Pi subunit resulted in a complete loss ofSNP stimulation.When Mn2+ was used as the cation, basal enzyme activities ofall mutants increased as expected, whereas, for unknownreasons, basal activity of the wild-type enzyme remainedunchanged. The (3H105F mutant exhibited basal enzymaticactivity amounting to halfofthe basal activity ofthe wild-typeenzyme and remained NO-insensitive. To ensure that nonre-sponsiveness toNO ofthe P3H105F mutant was not restrictedto stimulation by SNP, S-nitrosoglutathione (GSNO) (17),another NO-containing agent, was used to activate the wild-type and mutated enzymes. As shown in Table 2, the resultsobtained with GSNO were similar to those obtained with SNP.Determinations of intracellular cGMP levels in intact COScells transfected with the His-105 mutantfurther supported theinsensitivity toGSNO as incubation with the stimulatordid notincrease intracellular cGMP levels. Another activator of sol-uble guanylyl cyclase, protoporphyrin IX, which has beenshown to activate the enzyme in a NO-heme-independentmanner (18), failed to increase the catalytic activity of the3H105F mutant but led to a severalfold stimulation of thewild-type enzyme and of all other mutants (Table 2). Furtherevidence that His-105 of the A subunit is essential for NO-induced stimulation of soluble guanylyl cyclase was obtainedby the use of KCN, known to bind to the heme iron, therebyinhibiting the NO-induced stimulation of soluble guanylylcyclase (19). KCN inhibited both the NO-stimulated andnonstimulated wild-type enzyme to =30%6 of the originalnonstimulated activity. The remaining activity most likelyreflects the heme-independent catalytic activity ofthe cyclase,suggesting heme-dependent activation even in the absence ofadded SNP. In contrast, the activity of the 13H105F mutantremained unchanged as a function of KCN concentrations(Fig. 3).The insensitivity to KCN and the lack ofresponsiveness to

NO of the AH105F mutant could be due to a failure to bindheme or to a failure of the heme-containing enzyme totransduce the binding signal. To maintain the positive chargeofHis-105 that possibly represents the functionally importantfeature of this residue, basic amino acids (Lys and Arg) werealso substituted at position 105 (fH105K and 1H105R,respectively). Such a mutation (His -- Arg instead of HisVal) has been shown to at least partially restore enzymeactivity of the adenylyl cyclase of Bordetella pertussis (20).Both mutants were coexpressed with the unmodified a,subunit in COS cells, and the expression was verified on

2594 Biochemistry: Wedel et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 2595

c

E

0.ECO.c D200-C.)

LC)

C .CZo

(DEC 0-

2

KCN, mM4

FIG. 3. Influence of KCN on activities of the wild-type guanylylcyclase and of the PtH105F mutant. Enzyme activity was measuredin the cytosol of COS cells transfected with cDNAs of the al and Pisubunits with (solid circles) or without (open circles) 0.1 mM SNPand in the cytosol of cells transfected with thecDNA ofthe al subunitor the cDNA of the P3H1O5F mutant (diamonds). The assay wasperformed for 15 min at 3TC with 10 jsM GTP, 3 mM Mg2+, and theindicated KCN concentrations.

Western blots (data not shown). By taking into considerationthe pKa values of Lys and Arg, the determination of cGMPformation in the cytosol of the transfected cells was carriedout at various pH values (pH 7.5-9). Although activity of thewild-type enzyme was decreased in a pH-dependent manner,the enzyme still exhibited one-third and one-fifth of its basaland stimulated activities, respectively, at pH 9. In contrast,both mutants failed to demonstrate catalytic activity undernonstimulated or stimulated conditions (SNP or protopor-phyrin IX).

To determine whether the mutant still bound heme, the al

and (,1 subunits were expressed in Sf9 cells to obtain largerquantities of the enzymes. As in COS cells, the /,1H105Fmutant coexpressed with the al subunit in the insect cellsexhibited basal activity that was not stimulated by NO,whereas the wild-type enzyme was stimulated -60-fold. Im-munoaffinity chromatography with peptide antibodies di-rected against the 3,1 subunit as described by Humbert et al. (6)was used to purify the wild-type and mutant enzymes toapparent homogeneity as judged on SDS gels. Subsequently,

0.0040.0081

~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . 0

= 0.006

Sf9 wild type

C$0.004-

Q0 \ / His-105 mutantco

cdcla 0.002 0.001 °

.0Bovine lung

0 0

280 330 380 430 480Wavelength

FIG. 4. Absorbance spectra of purified wild-type and mutantguanylyl cyclases. The wild-type (4 gg) and the mutant (4.8 ug)enzymes purified from Sf9 cells and soluble guanylyl cyclase purifiedfrom bovine lung (4.8 ,ug) were applied to a Sephadex column. Theabsorbance from 260 to 600 nm was monitored by means of aphotodiode array detector.

absorbance spectra ofthe wild-type and mutant enzymes wereobtained. For comparison, soluble guanylyl cyclase purifiedfrom bovine lung also was analyzed (Fig. 4). The wild-typeenzyme and the enzyme purified from bovine lung had absor-bance maxima at 430 nm, typical for hemoproteins (Soretband), and exhibited similar A2o/A430- ratios, indicating com-parable amounts of enzyme-bound heme, whereas detectableabsorption at 430 nm for the PBH105F mutant was not ob-served. Based on the heme content ofthe lung enzyme (0.3 molof heme per mol of enzyme) (6) and the limits of detection,there is <0.03 mol of heme per mol of enzyme in the mutant.

DISCUSSIONCharacterization of several His mutants of soluble guanylylcyclase revealed that His-105 ofthe /31 subunit is essential forthe stimulation of soluble guanylyl cyclase by NO, as thereplacement of this residue with Phe yielded a catalyticallyactive enzyme that was not stimulated by NO. The PH105Fmutant continued to form dimers with the al subunit asdemonstrated by immunopurification of a heterodimeric en-zyme from Sf9 cells.

It has been proposed by others (4, 18, 21) that enzyme-bound heme is required for the stimulation of soluble gua-nylyl cyclase by NO and that activation is initiated by bindingof NO to the heme moiety. Our studies show a loss ofenzyme-bound heme in the mutant enzyme, and thus, thenonresponsiveness to NO is most likely due to a failure tobind heme; this is in agreement with the failure of protopor-hyrin IX to activate the mutant. The heme deficiency couldbe caused by a conformational change induced by the mu-tation that does not disrupt basal enzymatic activity. Alter-natively, His-105 may directly participate in heme binding(for example, by interaction with the propionic acid residuesof the heme moiety) or His-105 may, analogous to theproximal His residues of hemo- and myoglobins, form alinkage to the fifth coordination position of the heme iron,thereby stabilizing the heme-protein interaction. In supportof the latter proposal, His-105 is preceded by a Leu in the -4position (Leu-101) as are the proximal His residues of allvertebrate hemo- and myoglobin types known to date.

Although we can only speculate about possible mecha-nisms impairing heme binding in the modified enzyme, theP1H105F mutant demonstrates that the heme prostheticgroup is required for the stimulation by NO but is not requiredfor catalytic activity. The mutant, therefore, may serve as animportant tool in future studies on the heme dependency ofother known regulators of soluble guanylyl cyclase (19).His-105 is conserved in the 3 subunits and is not found in thea subunits, pointing to possible distinct functions of thesubunits with respect to heme binding. Future studies shoulddetermine whether the P, subunit is capable of binding hemeindependently of the a subunit. The mutant suggests that theenzyme-bound heme exists with the N-terminal region of thepolypeptide chain. Since the C-terminal domain has beenproposed by sequence homologies to represent the catalyticdomain, the N-terminal region may function as the regulatorydomain and, therefore, resemble in some respects the regu-latory N-terminal nature of the membrane-bound forms ofguanylyl cyclase.

We are indebted to Monika Bigalke for skillful technical assis-tance. We thank Dr. Vijay S. Sharma for helpful discussions. Thiswork was supported by the Deutsche Forschungsgemeinschaft andby the Fonds der Chemischen Industrie.

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