In early studies of cytochromes, spectroscopic methods were relied upon for determination of spin state, coordination number, and axial-heme ligation. Model hemes in which metal-ion size, charge, and d-orbital occupancy, as well as ligand structure and composition, could be system- atically varied, were spectrally characterized for comparison to cytochromes. Synthetic 6cLS FeII and FeIII porphyrinates have been studied for many years with the goal of clarifying structure–
function relationships observed in cytochromes.
Thestructures of many 6cLS FeIIand FeIIIporphyrinates are available.11 TheFeIIIcomplexes exhibit several characteristic structural properties. There is little or no displacement (0.09 A˚) of the FeIIIcenter from the mean heme plane, even in complexes with different axial ligands. The Fe–
Nprrole(FeNp) bond lengths depend upon the charge of the complex, with FeNpbeing1.990 A˚
for neutral or positively charged complexes, and 2.000 A˚ for complexes with a single negative charge.11 TheFeNp distances within a given complex can be inequivalent if axial ligands are coplanar and nearly aligned with the FeNp bonds (0,Figure3), thoseperpendicular to the ligand plane being shorter by0.02 A˚. Axial FeIIIL bond lengths depend upon orientation if the ligand is planar. Steric interactions of axial ligands like pyridines and hindered imidazoles with the porphyrin ring tend to lengthen the FeIIIL bond. Finally,-acceptor ligands can induce ruffling of the porphyrin core by electronic stabilization.
Equatorial FeNp bond lengths in the 6cLS FeII porphyrinates are only slightly longer (0.0102 A˚) than their FeIIIcounterparts, and in the complexes with identical axial ligands, the FeII atom is in theplaneof theporphyrin core.11 In theLS d6 configuration of theferrous cytochromes and model hemes, filling of thed orbitals is expected to favor increased-bonding in these complexes. This is borne out by shortening of bonds with -acceptor ligands by hundredths of angstrom relative to the corresponding FeIII complexes.11 In model hemes, the FeS bond lengths for neutral sulfur ligands appear to be insensitive to whether the iron is in the þ2 orþ3 oxidation state. Bonds to neutral nitrogen donor ligands such as imidazoles, pyrazoles, and pyridines exhibit longer bonds in the FeIIcomplexes.11
8.2.2.2 Out-of-plane Porphyrin Deformation
One of the more extensively studied and discussed structural variables has been equilibrium out- of-planedistortion of theporphyrin. Out-of-planeporphyrin deformation typically results in a bathochromic (red) shift of the optical absorption spectrum and a shift to more negative reduc- tion potentials (easier to oxidize).12–15These tantalizing correlations, based upon studies of model complexes comprising porphyrin ligands such as octaethyltetraphenylporphyrinate (OETPP2), having sterically crowded peripheries that force large out-of-plane deformations, have driven
N N
N N
or x
y
Xxx Xyy
φ γ0
γ
Figure 3 Heme core showing rotation angle of planar axial ligand(s) with respect to the Fe–Np(positivex) axis, or0. Also shown is the equivalent counterrotation, , of the magnetic susceptibility tensor axes.
Adapted from ref.27.
widespread investigation of the relationships between out-of-plane deformation, redox potential, and optical spectra of porphyrins and metalloporphyrins.12–14
Several reports have called into question the correlation between sterically enforced, out- of-plane deformation and the electronic properties, redox potential and spectroscopic signatures.16–18 Based on UV–visible spectra, structural analysis, and TD-DFT calculations, it has been suggested that the shifts in UV–visible transitions result from electronic effects of the peripheral substituents on the aromatic porphyrin core. These effects drive a bond-alternating rearrangement of the porphyrin core atoms called ‘‘in-plane nuclear reorganization.’’16
Regardless of the driving force(s), out-of-plane porphyrin deformations invariably occur along low-frequency, normal coordinates. A recent computational method, normal-coordinate struc- tural decomposition (NSD), yields linear combinations of equilibrium distortions along the lowest- frequency out-of-plane normal vibrational coordinates of the heme.15,19,22 Thesix out-of-plane vibrational coordinates of aD4hmetalloporphyrin used in the linear combinations are illustrated inFigure4. NSD results show that hemes in many cytochromes exhibit equilibrium out-of-plane distortions of nearly 1 A˚ along therufand/orsadcoordinates, and it has been estimated that these distortions require heme protein interaction energies substantially greater than 2 kcal mol11and up to 8 kcal mol11,23,24 Interestingly, out-of-plane deformations are largely conserved within classes of cytochromes.21,22,25,26
It has been suggested that expenditure of energy by a protein to enforce a particular out-of-plane distortion is unlikely to be accidental, and could play an important rolein biological function.15,23
8.2.2.3 The Frontier Orbitals and Fe–Ligand Bonding
Figure5 shows the nodal patterns of the four highest occupied and the two lowest unoccupied porphyrin molecular orbitals (MOs)27 and the energy ordering and symmetry designations of
sad[B2u], 65 cm−1 ruf[B1u], 88 cm−1
dom[A2u], 135 cm−1 wav[Eg(x,y)], 176 cm−1
pro[A1u], 335 cm−1
Figure 4 Six out-of-planevibrational coordinates of a D4h metalloporphyrin used in normal structural decompositions to reveal compositions of equilibrium out-of-plane heme deformations. Mulliken symbols areof theD4h point group and theabbreviations sad,ruf, dom, wav, and pro indicate saddled, ruffled, domed, wave, and propeller distortions, respectively. (Note that wavx and wavy derive from a doubly degenerate pair of Egvibrational modes of the D4h porphyrin.) Thedark and light circles indicateatoms
on opposite sides of the mean heme plane. Adapted from ref.11.
metal-centeredd-orbitals in octahedral and tetragonal ligand fields, along with the frontier MOs of the porphyrin. Sigma bonding between iron and the porphyrin is based on interactions between the dz2 (a1gin D4h) and dx2y2 (b1g in D4h) orbitals of iron and porphyrin MOs with respective symmetries. The degeneratedxz,dyzpair (eginD4h) can combinewith theporphyrinMOs ofeg
symmetry.27Thedxyorbital is nonbonding inD4hmetalloporphyrins. Of particular interest is the bonding interaction between the dxyorbital (b2symmetry) and the porphyrinate b2MO (correl- ates with thea2uMO in D4hcomplexes) in hemes ofD2dsymmetry.24,27–32Symmetry lowering to D2dis usually associated with the ruffling distortion shown inFigure4. This deformation involves counterrotation of adjacent pyrrole rings about their FeNp bonds, which results in a nonzero projection of pyrrole N pz-derived MOs onto the mean porphyrin plane. The resulting –dxy
bonding represents an electronic contribution to the stability of some ruffled 6cLS FeIIIporphy- rinates with (dxz,dyz)4(dxy)1 FeIII ground states. This stabilization is not possible for FeII com- plexes, because the dxyorbital is filled.
Sigma bonding between Fe and axial ligands generally involves 3dz2, 4s, and 4pzorbitals of the metal and occupied orbitals of the axial ligands. Axial ligands can also participate in bonding with FeIIIand FeIIcenters by interaction of theirorbitals with the partially or completely filled dxzanddyzorbitals.27
In most 6cLS FeIIIhemes, the ordering is that of the (dxy)2(dxz,dyz)3ground state.27This stateis subject to stabilization by Jahn–Teller splitting of the egset, which results in symmetry lowering from fourfold to twofold rotational symmetry. The stabilization due to Jahn–Teller splitting in 6cLS FeIII porphyrinates can manifest itself as experimentally discernible symmetry lowering, including the thermodynamic preference for parallel orientations of planar axial ligands and asymmetry in FeNp bond lengths when the ligands align with two opposing FeNp
bonds.11,25,33,34
Such complexes give rise to rhombic (type II) EPR spectra,27,28,35 as shown in Figure6. Although according to the Jahn–Teller theorem, dxz and dyzcannot be degenerate in
N N NFeN
N N N N
N N N N V
∆ dx2-y2; b1g
dz2; a1g
dxy; b2g dxzdyz; eg
dx2-y2; b1 dz2; a1
dxy; b2 dxzdyz; e
dx2-y2; ag dz2; ag
dxy; b1g dyz; b3g dxz; b2g eg
eg a1u a2u
e
e b1 b2
b2g
au b1u b3g
b2g b3g
dx2-y2; b1g dz2; a1g dxy; b2g dxzdyz; eg
dx2-y2; b1 dz2; a1 dxy; b2 dxzdyz; e
dx2-y2; ag dz2; ag
dxy; b1g dyz; b3g dxz; b2g eg
eg a1u a2u
e
e b1 b2
b2g
au b1u b3g
b2g b3g
D4h D2d D2h
E
E Strong axial
ligand field
Weak or π- accepting
axial ligand field Nodal patterns & symmetries
of D4h porphyrinate MOs 4eg; π*
LUMO
3eg; π 3a2u; π 1a1u; π HOMO
Figure 5 Nodal patterns of the four highest occupied and the two lowest unoccupied porphyrinateMOs are shown at the left (adapted from reference27). At theright areshown theirond-orbitals and porphyrin MOs, along with their symmetries in the D4h, D2d, and D2h point groups. Symmetry-allowed bonding/
antibonding orbital interactions are shown by dashed lines connecting porphyrinate and d-orbitals of the same symmetries. Heme structures are shown for each point group to illustrate the effect of axial ligand orientation on the symmetry of the molecule, whether or not the porphyrin experiences out-of-plane distortion. If the axial ligands are not identical, the heme has either C2v or lower symmetry. In these cases all of thed- and porphyirn-orbitals shown are allowed by symmetry to participate in bonding/antibonding interactions. The relative energies shown are arbitrary and are not intended to accurately reflect energy
match or mismatch of metald- and porphyrin-orbitals.
6cLS FeIIIhemes, the effects of Jahn–Teller splitting can be masked by strong steric interactions between the porphyrin and axial ligands, due either to out-of-plane deformation of the porphyrin or to sterically demanding axial ligands11such as 2-methylimidazole, which force the axial ligands to be mutually perpendicular. These complexes also have (dxy)2(dxz,dyz)3ground states and give riseto highly anisotropic (typeI or ‘‘gmax’’) EPR spectra.27,28,35In complexes with strong axial- ligand fields imposed by weak -donor and/or-acceptor ligands, such as 4-cyanopyridine and theorganic isocyanides, theMOs having contributions from thedxzand dyzAOs arestabilized.
These complexes have axial (dxz,dyz)4(dxy)1ground states,11,27 which arenot susceptibleto Jahn–
Teller distortion and exhibit type III EPR spectra.27 This ground state is further evidenced by substantial weakening of the porphyrin d (2B2!2A1) NIR-CT MCD transition, which is symmetry forbidden for (dxz,dyz)4(dxy)1ground states.28
8.2.2.4 Cause and Effect Roles of Axial Ligation
Planar axial ligands can adopt mutually parallel or mutally perpendicular orientations in 6cLS FeIII complexes, with their orientations relative to the FeNp bonds ranging from 0 to 45.27
(a)
(b)
(c)
(d)
3.32
2.90
2.33
1.52
2.57
2.21
1.93
[Fe(TMP)(4-NMe2Py)2]+
θ = 79°,φ = 73°
∆Cm = 0.51 Angst.
Fe–Np = 1.964 Angst.
Fe–Nax = 1.984 Angst.
[Fe(TMP)(NMeIm)2]+
θ = 0°,φ = 41°
∆Cm < 0.03 Angst.
Fe–Np = 1.984 Angst.
Fe–Nax = 1.970 Angst
[Fe(TMP)(4-CNPy)2]+
θ = 87˚,φ = 40˚
∆Cm = 0.41 Angst.
Fe–Np = 1.961 Angst.
Fe–Nax = 2.011 Angst.
[Fe(TMP)(t-BuNC)2]+
∆Cm = 0.38 Angst.
Fe–Np = 1.977 Angst.
Fe–Nax = 1.915 Angst.
0 100 200 300 400 500 600 700
Magnetic field (mT)
Figure 6 X-band EPR spectra of 6cLS ferric hemes. (a) Type I or ‘‘large gmax’’ spectrum typical of complexes with mutually perpendicular axial ligands. (b) Type II or normal rhombic spectrum typical of complexes with mutually parallel axial ligands. (c) Type III axial spectrum with large, and (d) small difference betweengIandgII. Type III spectra are typical of 6cLS complexes having axial-acceptor ligands
such as isocyanides. Figure adapted from reference27.
Relevant angles are shown in Figure3. There are two principal aspects of axial ligation to consider, steric and electronic. Planar axial ligands prefer mutually orthogonal orientations if FeL bonding causes steric interactions between the axial ligands and the porphyrin. These interactions are stronger for the six-membered pyridine and sterically hindered 2-methlyimidazole ligands than for imidazole.11,27 They can involve atoms in the porphyrin core and/or bulky peripheral substituents, and usually result in ruffling of the porphyrin core (see Figure4).
A distinct situation arises in persubstituted porphyrins, wherein strong steric interactions occur between adjacent substituents at the porphyrin periphery. These porphryins exhibit an inherent saddling deformation, even as metal-free macrocycles, and exhibit such narrow clefts that steric constraints dictate orthogonal, or nearly orthogonal, orientations of axial ligands.27,36 Most orthogonal axial-ligand orientations in 6cLS FeIII porphyrinates are attributable to the relief of costly intramolecular steric interactions.11,34,37–39
This is an important point with regard to the heme structures and deformations in cytochromes, as their hemes exhibit little intrinsic intramolecular steric strain.23 Furthermore, since all type b, c, and fcytochromes characterized to datehave(dxy)2(dxz,dyz)3 ground states,27,40,41 there is no electronic stabilization of the ruffling distortion as described above in these cytochromes. Hence, equilibrium deviations from planarity must be attributable to exogenous forces brought to bear on the heme by the protein.
In the absence of steric encumbrance at the porphyrin periphery, the parallel axial-ligand conformation is favored for ligands such as unhindered imidazoles. This is attributed to stabiliza- tion of the Jahn–Teller distorted ground state.11,27,34 In these complexes, the porphyrin is invariably flat and , theligand angle, can rangefrom 0 to 45 (Figure3). The complexes havingnear 0 exhibit rhombic EPR spectra and aB1g-distorted porphyrin core (nonequivalent adjacent FeIIINp bond lengths). Those with near 45 exhibit equivalent FeIIINp bond lengths, but still yield rhombic EPR spectra wherein the rhombicity, V (see Figure5), has been shown to track inversely with.42Six-coordinate, low-spin FeIIcomplexes strongly prefer parallel axial ligands and a planar porphyrin ligand.11,27,29This is thought to bebecauseof theinability of low-spin FeIIto stabilize a ruffled porphyrin conformation. Several examples of ruffled 6cLS FeII complexes have been reported.43 Ruffling has been ascribed to steric interactions between bulky porphyrin substituents and axial ligands. Some work has suggested that FeNax bonding is independent of -accepting ability of the axial ligands; these results were interpreted to mean thatdxz- anddyz-orbitals arenot strongly involved in-bonding.29Consistent with this reasoning, a series of 4-substituted pyridines with varying pKas and -acceptor abilities show the same thermodynamic stabilities, suggesting that axial bond strengths do not vary significantly in this series. This is in contrast to the analogous FeIIIcomplexes, wherein the FeNax bond strength varies predictably.44 Since the redox potential depends upon the ratio of FeIIIand FeIIstability constants, log(2III
/2II
),44 tunability of the contribution of bond strengths to redox potential seems to be confined to tunability of 2III in model complexes. It is worth noting that this may not be the case in cytochromes, because the proteins have control over porphyrin conformation and axial-ligand orientation, which can differ from the lowest-energy conformations of the model complexes where there are no exogenous forces.
8.2.3 CYTOCHROMESc 8.2.3.1 Function
Cytchas an important role in the production of ATP; in the mitochondrial respiratory electron- transfer chain, cytctransfers electrons from the transmembrane cytbc1complex to cytochromec oxidase.45,46 Cyt c also delivers electrons to cytochrome c peroxidase, which facilitates the reduction of hydrogen peroxide to water. In addition to its life-sustaining electron-transfer functions, cyt cis required for activation of the cell-death protease, caspase-3, in apoptosis.47–49 Defects in cyt cbiogensis have been implicated in pathogenic responses related to copper50 and iron metabolism,51and prokaryotic heme biosynthesis.52
Similarc-type cytochromes are involved in many kinds of energy metabolism in bacteria, such as phototrophes, methylotrophes, sulfate reducers, nitrogen-fixers, and denitrifiers. For example, in the anaerobic electron chain of the denitrification system in Gram-negative bacteria, c-type cytochromes transfer electrons from the cytbc1complex to cytochromecd1nitrite reductase, N2O
reductase, and NO reductase.53In plants and cyanobacteria,c-type cytochromes shuttle electrons from thecytb6fcomplex to photosystem I.54
Dueto its central rolein thevital processes of living organisms, and thelargedatabaseof physical and biochemical information available on cyt c, it has becomeoneof theparadigms in the study of biological electron-transfer processes.55,56 Functional studies on cyt c and numerous cytcpoint mutants have been used to identify residues and regions of the protein that influence electron-transfer properties.55–57In order to study intraprotein electron transfer and thepathways involved in cytc, numerous donor–acceptor complexes have been generated by covalently linking various redox-active inorganic complexes to surface amino-acid residues of cytc.58–60Interprotein electron transfer has also been examined using cytccomplexes with cytochrome c oxidase,61–65 plastocyanin,66–69 cyt b5,70–72 and cytochrome c peroxidase.73–76 Sincecytcis ubiquitous, easy to isolate, stable, and soluble, it has also become a system for thestudy of protein folding77–86 and protein dynamics.87–90
8.2.3.2 General Classifications
Cytscfall into at least four general classes.5,91,92Thelargest class, class I, consists of small (8–
20 kDa), monoheme cytscthat arehomologous to mitochondrial cyt c. Sequence and structu- ral data have be e n use d to divide class I into sixte e n subclasse s.92 Mitochondrial cyts c and purplebacterial cyts c2 makeup thelargest subclass. Additional subclasses includePseudo- monascyts c551, cytsc4, cytsc5, cytsc6(algal cytsc553),Chlorobium cytc555, Desulfovibriocyts c553, cyanobacterial and algal cyts c550, Ectothiorhodospira cyts c551, flavocytochromes c, methanol dehydrogenase-associated cyt c550 or cL, cytochrome cd1 nitrite reductase, alco- hol dehydrogenase and its associated cytochrome subunit, Pseudomonas nitritereductase- associated cytc,Bacilluscytc, andBacilluscytochromeoxidasesubunit II. With thelargenumber of cyts c presently being characterized structurally, proteins that fall into new additional subclasses continue to be found.93,94 Most class I cyts c arewater solubleand contain a 6cLS heme. Their heme-attachment site (–Cys–Xxx–Yyy–Cys–His–) is towards the N-terminus.
The axial heme iron ligation is provided by the His residue of the heme-attachment site and a Met residue found near the C-terminus.Figure7(a) illustrates the general fold for class I cyts c.
Class II cytschavea singlec-type heme covalently linked to the highly conserved –Cys–Xxx–
Yyy–Cys–His– sequence near their C-termini.5 While the number of conserved amino-acid residues among class II cytsc is relatively small,5,95 the structural motif of a left-twisted, four- -helix bundle is characteristic of these proteins (Figure7(b)).96,97 A His residue occupies one axial coordination site of the heme iron, while the second axial coordination site is variable. This class has two subclasses that are distinguished by the spin state of the heme. Subclass IIa consists of cytsc0, which havehigh-spin (HS) configurations in thereduced form [FeII,Sẳ2] and either HS (Sẳ5/2)98–101 or a quantum-admixed spin (admixture ofSẳ5/2 andSẳ3/2) states98,100,102–104 in the ferric form. For example, the observed g-values for ferric Chromatium vinosum cyt c0 (g1ẳ5.32, g2ẳ4.67,g3ẳ1.97) are not typical for a purely high-spin ferric heme, and simulation of this EPR spectrum reveals that the electronic ground state of this cytc0consists of 51%Sẳ3/2 and 49% Sẳ5/2.100 These cyts c0 are found in photosynthetic and denitrifying bacteria.
Subclass IIb includes proteins like cytc556 from Rhodopseudomonas (Rps.)palustris,105 Rhodo- bacter (Rb.)sulfidophilus106 and Agrobacterium tumefaciens,5 and cyt c554 from Rb. sphaeroides107 that contain low-spin hemes. In these cases, the sixth heme ligand, a Met, is found near the N-terminus.
Triheme, tetraheme, octaheme, nonaheme, and 16-heme cytscfrom sulfateand sulfur-reducing bacteria are included in class III.5,92,108–112
Gene duplication of tri- and tetraheme units is clearly apparent. In Desulfovibrio species, the tetraheme proteins (15 kDa) are part of the electron- transfer chain that couples theoxidation of molecular hydrogen by hydrogenaseto sulfatereduc- tion.113 These multiheme proteins, also known as cyts c3, generally have bis-His heme ligation.
Although the conserved amino acids in this class appear to be limited to the heme-binding cysteines and the heme iron axial histidines, the three-dimensional structures ofDesulfovibrio tetraheme cyts c340,114–121 reveal that the overall protein fold with two short -strands and 3 to 5 -helices is conserved.92 Interestingly, the heme–heme distances and heme–heme angles are evolutionarily highly conserved. The hemes are in close proximity to one another, with adjacent pairs of heme planes nearly perpendicular to one another (Figure7(c)). There is, however, considerable variability in the dihedral angle between the axial His planes inDesulfovibrio (D.)cytsc3.119,120,122
Class IV consists of large (40 kDa) tetraheme photosynthetic reaction center (THRC) cyts c.
Rsp. viridis THRC cyt c is the electron donor to the bacteriochlorophyll special pair. The structure of this membrane-associated Rsp. viridis cytc exemplifies the tertiary structure of this class (Figure7d).5 Its four hemes are in a linear arrangement, with alternating high- and low- potential sites . WhileRsp. viridisTHRC cytchas three hemes with bis-His ligation and one with His/Met ligation, the homologous THRC cyts cfrom Chloroflexus aurantiacus92and Rubrivivax (Rv.)gelatinosus123appear to contain only His/Met heme ligation.
Nitrosomonas (N.)europaeacytc554is a tetraheme that is not homologous to either class III or class IV multiheme cytochromes, and is considered to be in a class of its own (Figure7e).
Involved in the biological nitrification pathway, this cyt c554accepts two electrons from hydro- xylamine oxidoreductase (HAO) upon generation of nitrite. Its one high-spin heme and three 6cLS hemes (þ47, þ47, 147, and 276 mV vs. SHE) are arranged in two types of pairs where the hemes are in van der Waals contact. Hemes III/IV have their porphyrin planes nearly perpendicular to one another in an arrangement similar to that in cyts c3.6 Heme pairs I/III and II/IV have nearly parallel porphyrin rings that overlap at one edge, similar to the heme arrangement in HAO and cytochrome c nitrite reductase.6 Sequence similarities between these seemingly unrelated proteins are found in the polypeptide near the hemes when the heme-stacking arrangement is used to align the protein chains.124Based on this and the conserved nature of the heme organization, it has been suggested that N. europaea cyt c554, HAO, and cytochrome c nitrite reductase have a common evolutionary origin, but have diverged to fulfill different functions.
A sixth class ofc-typecytochromes consists of thecytsffrom thecytb6fcomplex of oxygenic photosynthesis. The crystal structure cytfon the lumin-side reveals two elongated domains made up primarily of-sheet secondary structure, with the heme attached to the larger domain close to
Met 80 Met 80
His 18 His 18
His 26 His 26
His 33 His 33
His 39 His 39
Lys 73 Lys 73
Lys 79 Lys 79
(a) (a)
(b) (b)