5515
J . Am. Chem. Sac. 1994,116. 5515-5516
Vibrational Characteristics of Mutant and Wild-Type Carbon Monoxy Cytochrome e Oxidase: Evidence for a Linear Arrangement of Heme a, ah and CUB Jonathan P. Hosler,' Younkyoo Kim.tl J. Shapleigh,~~v R. l3ennis.c J. Albcn,l S. Ferguson-Miller.' and G. Babcock'.: Departments of Biochemistry and Chemistry and LASER Laboratory. Michigan S t a t e Uniuersity East Laming, Michigan 48824 Department of Biochemistry University of Illinois. Urbana, Illinois 61801 Department of Medical Biochemistry Ohio S t a t e University. Columbus, Ohio 43210
Received January 13. 1994 The combined application of genetic, biochemical. and spectroscopic techniques to bacterial versions of the key oxygenmetabolizing enzyme of aerobic organisms. cytochrome oxidase, has led to topological models of the heme and copper cofactors within the protein framework.l-' The current view places the oxygen-activating center, heme a l l C u ~and , the low-spin heme, hemea,withinsubunit loftheenzyme. Inthemostrecent model (Figure I ) , both hemes are bound to transmembrane helix X, with His102 and His421 functioning as the ligands for heme a and His419-ligating herneal(seerefs I and2). Threeremaining conserved histidines, His284, -333,and -334,areassigned as CUB ligands. A polypeptide loop located on the outer side of the membrane connects helices IX and X and contains a number of conserved residues, including Asp412. According to the model, Asp412 is on the proximal side of heme a,, while His333 is on the distal side, as a ligand of CUB.Although there is substantial evidence to support these metal ligand assignments.'" there have been some conflicting data regarding the designation of His419 or His284 as the ligand of heme This assignment dictates whether Cus is located between or distal to the two hemes, and thus is critical for understanding its function.'.8 Herewe havetested themodelofFigure I byanalyzingmutants a t the 333 and 412 positions for their vibrational properties in both the unliganded fully reduced and carbon monoxy forms. Mutants in cytochrome aa, of Rhodobactersphaeroides in which His333 or Asp412 of subunit I was substituted with asparagine (His333Asn and Asp412Asn. respectively) were constructed as
' Dcpnmcnt of Biochemistry. Michigan Slate University 1 Depanmcnl of Chcmirrr)
and the LASER Laboralory. Wichigan Slate
university I Department of Biochemistry. University of Illinois. I Department of Medical Biochemistry. Ohio State University. I Praentaddrers: DepartmcntofChcmistry. Hankuk UnivmityofForcign Studies. ~. Yominsun. Kvunnki-do.449-791. Korea. Present Zd&: 'D;pa&ntaf Micrabiblogy. Cornell University. Ithaca. New Y a k . ( I ) Harler.J. P.;Shapleigh.J.P.;Tsklenburg.M. M. J.;Thomas.J. W.; Kim. Y.; Erpc. M.; Fcttcr. 1.; Babmck. G. T.; Alben. J. 0.;Gcnnir, R. E.; Ferguron-Miller. S . Bioehcmisrry 1994. 33. 1194-1201. (2) Harlcr. J. P.;Fcrguwn-Miller. S.;Calhoun. M.; Thomas, J. W.; Hill. 1.;Lemieur. L.; Ma. 1.;Georgiou. C.; Fetter,J.;Shapleigh.J. P.;Tecklcnburg. M. M. J.; Eabmck. G. T.; Gennir. R. E. J. Biwnerg. Biomcmbr. 1993. 25. 121-136. ~~~~
heme a
Flyre 1. Helical wheel model of five transmembranehelica ofsubunit I of Rhodobaeicrsphaeroides cytochrome eoxidase. showing thepropmcd location of the three metal centers and an extramembraneloop that caps the heme a, heme a]. and Cue metal centers. The histidine ligands of the metabare numbered according tothe Rb.sphacroidcssaluenceand shownassolidblackcircles;otherwnsclvcdresiduesin the transmembrane helices are shown in bold circles. The two residues that are the focus of this study arc in black boxes
8 H333N w
W.T.
r
z
I
+
+
I2CO
'IC0
I
390
650 WAVENUMBERS
Rgne 2. Resonance Raman spMra of CO-bound forms of wild-typs Rb. rphaeroides cytochrome e oxidase and mutant forms Ao~dl2Asn (D412N) and His333Asn (H333N). as indicated. The spxtra were obtained at room tsmprature with 5-cm-I resolution by using a Spx 18778triplespxtrographandan EGBG Model 1421diodcarraydetstor. The oxidax samples (30-50 rM) were placed in a spinning cell and excited at424 nm witha Coherent 590dyelaserwnnaedwithaCoherent lnnova 200 argon laser. Laser power at the sample was less than 2 mW, and spectra were c o l l a e d within 5 min of initiating laser irradiation. ' T O was added to the r e d u d enzyme by repeated evacuation and flurhingwithC0, T O (CambridgeIsotopeLaboratories)wasexchanged for ' T O by the same method.
previously descriW;lJ the mutant enzymes were purified as in Hosler et al.9 Figure2 shows resonanceRaman spenra in thelow-frequency (3)Shap!cigh.J.P.;Halcr,J.P.;Tsklenburg,M.M.J.:Kim.Y.;Babmck. region for the carbon monoxy complexes of the mutant and wildG.T.;Genna. R. E.: Fcrguwn-Miller.S. Proe. Nor/.Acod.Sci. US.A. 1992. type oxidases. The stretching vibration of the iron-carbon bond 89,47864790. (4) Lemieua. L. J.;Calhoun, M. W.;Thomas,J. W.; Ingledew, J.;Gennis. of heme a3,v ( F b C 0 ) . is identified a t 5 16 cm-' for the wild-type R. E. J. B i d . Chem. 1992. 267.2105-2113. enzyme. This is confirmed by the 5-cm-' downshift in the 'IC0 ( 5 ) Minagawa. 1.; Mogi. T.;Gennis, R. B.: Anraku. Y . J. B i d . Chcm. isotope-labeling experiment, in agreement with the earlier work 1992.267. 20962104. (6) Calhoun. M. W.; Thomas, J. W.; Hill, 1.1.; Haler. J. P.; Shapleigh. by Rousseau and co-workers.lO In His333Asn. u ( F 4 0 ) is J. P.; Tecklenburg. M. M. 1.; Fergusan-Miller. S.; Babcock,G. T.:Alben. J. (9) Haler. J. P.: Fetlrr.I..Tsklcnburg. M. M.J.; bpe. M.: Lerms.C.; 0.; Gennir. R. E. Biochcmisrry 1993. 32. 10905-10911. Ferguron..Uillcr. S . 1. BJol.Chem 1992. 267. 24264-24272. (7) Babmck. G. T.; Wikstrom. M. Nnrvre (London) 1992.356.301-309. (IO)Argads. P V.; Ching. Y C ; Rourxau. D. L. S c i r m 1984. 225. (8) Lemon. D. D.;Calhoun. M. W.; Gcnnis. R. E.; Wmdruff. W. H. 329-331. Biochirnirfry1993.32. 11953-11956.
ooO2-7863/94/1516-5515$04.50/0
0 1994 American Chemical Society
Communications to the Editor
5516 J. Am. Chem. Soc., Vol. 116, No. 12, 1994
Table 1: Vibrational Data for Mutant and Wild-type Cytochrome c Oxidases" enzyme
v(FeNhh) (cm-1)
v(Fe-CO) (cm-1)
v(Cm)a3 (cm-1)
v(C=o)Cu~ (cm-1)
wild-type His333Asn Asp412Asn
214 214 218
516 500 516
1964 1964 1966
2060 2080 2060
a The v(Fe-Nfi,) vibrational results are taken from refs 9 (wild-type), 3 (His333Asn), and 1 (Asp412Asn). The v ( m ) data for wild-type and Asp412Asn are taken from refs 14 and 1, respectively.
I
1900
~
1940
I
~
I
1980
'
I
2020
~
I
'
2060
I
'
I
~
2100
Wavenumber Figure3. Low-temperature (10 K) FTIRdifference spectra (light minus dark) of membrane bound, CO-ligated wild-type Rb. sphaeroides cytochrome c oxidase and His333Asn (H333N). Conditions are as described in Shapleigh et aI.l4 The (Y and fl forms14 of v ( m ) , , and v(C=0)cuB are labeled where they can be identified. The peak at 2065 cm-1 in the spectrum of His333Asn membranes arises from the (Cu) stretch of a cb-type cytochrome c oxidase that is overexpressed in mutants containing an inactive aa3-type oxidase.lJ4 The C m (Fe) stretching mode of this enzyme underlies the fi form of u(-)", of the aa3-type oxidase.
shifted down by 16 cm-I to 500 cm-1, while in Asp412Asn, u(Fe-CO) is the same as that for the wild-type.ll These two mutants werealso studied by low-temperature FTIR, providing the stretching frequencies for CO bound to heme a3, v(-),,~, as well as to CUB,u(C%o)~,,. As previously described for Asp412Asn,' u(C=0)cun is the same as that for wild-type, while ~(CEO),, is slightly shifted to 1666 cm-I. The FTIR difference spectrum for CO-bound His333Asn is compared to wild-type in Figure 3. In the wild-type spectrum, the negative peak at 1964 cm-* and the positive peak at 2060 cm-' represent the stretching frequencies for the (Y forms of CO bound to heme a3and CUB,respectively. (@forms are also observed; see ref 14.) In His333Asn, u(C=O),, is unchanged, although the signal is slightly broadened. In contrast, u(C=0)cun is dramatically shifted to 2080 cm-1, and the signal is attenuated. Thus, in His333Asn, CUBshows less CO binding with a stronger carbonoxygen bond, indicating an altered environment that may weaken the copper
