Viewpoint Cite This: Biochemistry XXXX, XXX, XXX-XXX
pubs.acs.org/biochemistry
Hop to It Jillian L. Dempsey*,† and Matthew R. Hartings*,‡ †
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, United States Department of Chemistry, American University, Washington, DC 20016, United States
‡
states for their biochemical reactions must walk a fine line with respect to how these states are achieved. Their electron transfer partner (whether that is a small molecule redox partner or another protein) must itself be oxidizing enough to activate the enzyme. However, if this partner is too oxidizing, electron transfer becomes inefficient. In their paper Hole Hopping through Tryptophan in Cytochrome P450, Ener, Gray, and Winkler1 study electron transfer processes in engineered P450 variants. The authors, in collaboration with Cheruzel, have previously shown that it is possible to generate the high-oxidation state, catalytically active intermediate of CYP102A1 (a P450 variant) by exploiting a tethered photosensitizer,4 but there was something funny about this initial observation: electron transfer from the porphyrin to the photogenerated oxidant is 3 orders of magnitude faster than what is predicted on the basis of ΔG° and the 20.8 Å separating these two redox sites. The authors hypothesized that the tryptophan (W96), positioned between the oxidant and the heme center, allowed a hopping mechanism but had no evidence that this was the case. To test their idea, Ener, Gray, and Winkler turned to another P450 variant (CYP119) that displays a histidine in the position analogous to CYP102A1’s W96. Then they mutated the suspicious tryptophan in CYP102A1 to a histidine and similarly mutated the histidine in CYP119 to a tryptophan. With these four P450 variants, the authors confirmed that the intervening tryptophan helps to overcome the long electron transfer distances by providing a hopping site between the ruthenium complex and the heme (Figure 2). In CYP102A1, the photoinduced oxidation is turned off when the tryptophan is removed and turned on in CYP119 when the redox-active tryptophan is installed. In addition, the authors show that, despite non-optimal electron transfer driving forces (−ΔG° < λ), the presence of the tryptophan ensures rapid oxidation of the heme. Through these experiments, the authors have taken another step to clarify the mechanisms for electron movement within proteins. Certainly, the observation of hopping through tryptophan is another welcome observation of aromatic amino acids mediating rapid electron transfer. As nature is well aware, several short, non-optimal hops are better than a single optimized leap any day of the week. Through studies like this report, we will deepen our understanding of how nature balances electron transfer driving force and distance with singlestep or multistep pathways to ensure rapid long-range electron transfer with minimal loss of free energy. Finally, as Gray and Winkler have shown, multistep electron transfer pathways are not only essential for productive chemistry. They can also be
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s the details of enzyme mechanisms are revealed, the properties that make or break function can often seem more physical than biochemical. An ensemble of vibrations, a hydrogen bond with just the right length, or a properly placed tryptophan can make all the difference. A recent report from Ener, Gray, and Winkler1 is an example of this depth of understanding and speaks to the tools that nature uses to move electrons from one place to another within a protein. Rapid and efficient charge transport in redox enzymes is essential for biological energy transduction pathways. In many systems, multistep electron tunneling architectures facilitate electron flow across significant distances with minimal loss of free energy.2 Aromatic amino acids like tyrosine and tryptophan play critical roles in these multistep mechanisms. For example, in Photosystem II, the highly oxidizing P680•+ formed upon photoexcitation is responsible for oxidation of the oxygenevolving complex (OEC). Nature has carefully positioned a tyrosine residue between the electron accepting P680•+ and the OEC, which acts as a way station for the electron during its cross-protein journey. Similarly, in ribonucleotide reductase, a tyrosine radical initiates oxidation of a cysteine residue some 35 Å away to form a radical that promotes the production of DNAs. A conserved series of tryptophan and tyrosine residues along the electron transport path are thought to participate in the charge migration process. In both examples, electrons bypass the direct route between the two end points to move through a more circuitous pathway with stopping points between them. Anyone who travels knows it is faster to take a direct flight than to have a layover; how is it that nature does not play by the same rules? Extensive work mapping long-range electron tunneling across a variety of protein scaffolds has revealed that the electron tunneling time scales have an exponential dependence on distance in these media.3 In simple terms, it is faster to transfer an electron across two 10 Å hops than a single 20 Å jump, just as one crosses a stream using stepping stones rather than leaping the full distance (Figure 1). Because electron transport time scales of microseconds to milliseconds are necessary for these biological redox machines to function properly, electrons prefer to “hop” across long distances rather than tunnel in a single step and redox-active amino acids provide the critical hopping points that are necessary, acting as intermediate electron acceptors and donors along the route. Along with the electron transfer pathway, nature must also consider the energetics of the donor and acceptor in their oxidized and reduced states. Specifically, the most pertinent factors are the free energy of the redox reaction (ΔG°) and the energy that is required to rearrange atoms as a result of the electron transfer (λ). Semiclassical electron transfer theory states that the rate of electron transfer is fastest when −ΔG° is equal to λ. Enzymes whose active sites utilize high oxidation © XXXX American Chemical Society
Received: September 23, 2017
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DOI: 10.1021/acs.biochem.7b00950 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. I told you that you should have hopped! Carefully positioned tryptophan residues offer redox enzymes the opportunity to exploit multistep electron tunneling mechanisms to rapidly and efficiently transport electrons across long distances. Image credit: Erika Hartings. (2) Dempsey, J. L., Winkler, J. R., and Gray, H. B. (2010) Protoncoupled electron flow in protein redox machines. Chem. Rev. 110, 7024−7039. (3) Gray, H. B., and Winkler, J. R. (2005) Long-range electron transfer. Proc. Natl. Acad. Sci. U. S. A. 102, 3534−3539. (4) Ener, M. E., Lee, Y.-T., Winkler, J. R., Gray, H. B., and Cheruzel, L. (2010) Photooxidation of cytochrome P450-BM3. Proc. Natl. Acad. Sci. U. S. A. 107, 18783−18786. (5) Gray, H. B., and Winkler, J. R. (2015) Hole hopping through tyrosine/tryptophan chains protects proteins from oxidative damage. Proc. Natl. Acad. Sci. U. S. A. 112, 10920−10925.
Figure 2. A structural model of RuC97-CYP102A1 reveals that a tryptophan is positioned midway between the photogenerated oxidant and the porphyrin. Figure reproduced with permission from ref 1. Copyright 2017 American Chemical Society.
installed as an escape hatch for proteins that use highly oxidizing active sites to ensure that no structural damage occurs when oxidizing equivalents make their way to active sites but fail to find a substrate with which to react. In these cases, tryptophan and tyrosine residues can funnel electrons and holes away from vulnerable amino acids to mitigate damage.5
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Jillian L. Dempsey: 0000-0002-9459-4166 Matthew R. Hartings: 0000-0003-0658-939X Funding
J.L.D. acknowledges support from a Packard Fellowship in Science and Engineering and a Sloan Research Fellowship. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Ener, M. E., Gray, H. B., and Winkler, J. R. (2017) Hole Hopping through Tryptophan in Cytochrome P450. Biochemistry 56, 3531− 3538. B
DOI: 10.1021/acs.biochem.7b00950 Biochemistry XXXX, XXX, XXX−XXX
