Oxiforms: Unique cysteine residue‐ and chemotype‐specified chemical combinations can produce functionally‐distinct proteoforms

A single protein molecule with one or more cysteine residues can occupy a plurality of unique residue and oxidation‐chemotype specified proteoforms that I term oxiforms. In binary reduced or oxidised terms, one molecule with three cysteines will adopt one of eight unique oxiforms. Residue‐defined sulfur chemistry endows specific oxiforms with distinct functionally‐relevant biophysical properties (e.g., steric effects). Their emergent complexity means a functionally‐relevant effect may only manifest when multiple cysteines are oxidised. Like how mixing colours makes new shades, combining discrete redox chemistries—colours—can create a kaleidoscope of oxiform hues. The sheer diversity of oxiforms co‐existing within the human body provides a biological basis for redox heterogeneity. Of evolutionary significance, oxiforms may enable individual cells to mount a broad spectrum of responses to the same stimulus. Their biological significance, however plausible, is speculative because protein‐specific oxiforms remain essentially unexplored. Excitingly, pioneering new techniques can push the field into uncharted territory by quantifying oxiforms. The oxiform concept can advance our understanding of redox‐regulation in health and disease.


INTRODUCTION
Combinatorial complexity is powerful. Take genetics. 4-nucleotides can be combined in 256 ways to make one tetranucleotide. In a prime example of emergent complexity, tetranucleotide combinations encode genomes. The human genome harbours the instructions to post-translationally controlling protein function. [5] Like nucleotides and genomes, a relatively simple event-a reduced or oxidised cysteine-produces the emergent, hierarchal layers of complexity that govern elemental redox-regulatory phenomena:

Compositional defines what proteins can be oxidised-identity-
and to what extent-a protein molar abundance sensitive parameter. [6] 2. Spatiotemporal defines the properties setting protein composition in space and time within cellular nanodomains from organelles to biomolecular condensates. [7,8] Combining the same building blocks in different ways yields complex non-uniform space-geometries evolving on divergent temporal trajectories. [9] 3. Chemical defines the precise cysteine sulphur chemotype [10,11] (e.g., a disulphide bond). They cement the non-binary nature of cysteine oxidation and endow it with disparate chemotype-specific chemistries. Chemotype diversity echoes the plurality of (1) heterogenous ROS cysteine reacts with, [4] (2) direct and indirect cysteine oxidation mechanisms [12] and (3) associated regulatory phenomena. [11] All of the above (e.g., ROS-type), and hence chemotypes are shaped by 1 and 2.
Like the particle zoo, ROS defines a chemical menagerie of molecules with unique biophysical properties. [13] Echoing ROS diversity, a single protein molecule can adopt a plurality of cysteine residue-and chemotype-specified proteoforms. Like ROS, the protein moniker subsumes a myriad of unique proteoforms. For example, p53 (UniProt: P04637) can form 8.5 × 10 9 unique cysteine residueand chemotype-specified proteoforms. I wish to term the cysteine residue-and chemotype-specified proteoforms 'oxiforms' to capture the essence of the idea by combining 'oxi' from oxidation and 'forms' from proteoforms. [14] Herein 'oxiforms' refers to cysteine residue-and chemotype-specified proteoforms as opposed to any other redox proteoform (e.g., a single-molecule with a 3-nitrated tyrosine residue) and chemotype defines different post-translational cysteine modifications (e.g., a sulfenic acid). Along with other factors (e.g., splice variants), oxiforms comprise part of the wider proteoform landscape [15] Here I critique the emergent higher-dimensional combinatorial chemistry-dependent single-molecule complexity of oxiforms. Just as discrete genes emerge from combining different tetranucleotides, the essential idea I wish to express is that: cysteine residue and chemotypespecified combinations endow oxiforms with distinct chemistrydependent functions. The present discourse will (1) define oxiforms terminologically and mathematically; (2) provide a chemical basis for combination-dependent functions and speculate on their biological significance (e.g., for phenomena like bet-hedging [16] ) and (3) critique technologies for quantifying oxiforms.

PART 1. DEFINING OXIFORMS
I define an oxiform as the unique cysteine residue and chemotypespecified proteoform a single-molecule occupies. The number of unique oxiforms a single-molecule can occupy can be calculated by solving n R where n and r denote the number of cysteine sulphur chemotypes and residues, respectively. The chemical logic of n is specified by the exact sulphur chemotype integer. To a broad approximation, chemotype diversity [10,11] yields n = 22. Specifically, (1) reduced with (RS-/RSH), (2)  3-hydropropinic acid, [17] (21) sulfinic acid (RSO 2 ) and (22) sulfonic acid (RSO 3 ). N expands if all adducts from acrolein to itaconate or RSSH space (e.g., persulfide radical) are counted. [18,19] Separate intermolecular (e.g., lysine for NOS bridges [20] ) or intramolecular partners can multiply n by orders of magnitude owing to their proteome-wide scale.

PART 2. FUNCTIONAL SPECULATIONS
Mixing primary colours produces a kaleidoscope of spectrally distinct shades. Combining different chemistries in specific ways produces unique oxiforms (see Figure 2). To draw an analogy to paint, chemistries are the primary colour palettes and oxiforms are the resulting spectrum of shades. The underlying logic rests on the distinctive chemical properties of the cysteine sulphur chemotype 'colours' . An essential pre-requisite for oxiform 'shades' . Even if they were monochrome, adding or subtracting a chemotype over r would darken or lighten the shade. The word chemotype betokens specific chemistry. Chemotype-specific chemistries are unequivocally established. For example, RSOH is a soft electrophile and weak nucleophile compared to RSH. [10] And RSS − is a stronger nucleophile than either. [22] Chemotypes endow the modified sulphur atom with distinct steric, electrostatic, nucleophilic and hydrophobic characteristics; which often produce a structural effect. [23] They paint a chemical picture.
Depending on the molecular context (e.g., protein molecule and cysteine residue), chemotypes can produce the same, in uniform or different ways, or a unique, even diametrically opposed, functional effect. First, in cases where the active site relies on deprotonated sulphur nucleophile chemistry (e.g., C215 in the tyrosine phosphatase PTP1B [24] ), any chemotype masking the nucleophilic character will inactivate the enzyme. Ergo RSOH, RSSG and RSNO all inhibit PTP1B.
Less-appreciated C215-RSH is inhibitory. Despite a low C215 pK a some PTP1B molecules will inhabit the RSH oxiform. Second, unique chemistries produce distinct functional effects even on the same cysteine residue. Take EGFR: C797-RSOH activates it whereas C797-RSSG inhibits it. [25] There might be more examples lurking in the 500 cysteine residues that the Qian group [26] demonstrated to be modified by multiple distinct chemotypes. Pursuant to the foundational principle of redox-regulation [5] : chemotype-specific chemistry can regulate protein function.
Oxiforms are holistic entities. Holism means the totality of the n of each r matters. Consider a purely hypothetical example of a protein called X (PX) with three cysteines. Suppose two PX molecules were defined by different oxiforms RSH (PX-a) and RSSG (Px-B). Compared I wish to speculate on the functional significance of oxiforms. With ∼10 3 cysteine containing proteins, a single cell (SC) may possess 10 9-12 unique oxiforms. Their proteome and ROS differences, [28,29] means the abundance of many oxiforms will vary between cells. They host a perpetually evolving amalgam of co-existing oxiforms. Some may contain rare oxiforms. Their stochastic nature brings an arresting thought to mind: some oxiforms may only ever exist once. Or never. Oxiforms may also contribute to a defining feature of life: the inherent adaptability of cells. They can generate evolutionary significant bet-hedging behaviours [16] by enabling cells to mount divergent responses to the same stimulus. The ability to overtly to subtly perturb the function of a protein by generating unique oxiforms is evolutionary significant. The hotbed of functional possibilities may even enable certain favourable oxiforms to be selected for. Sometimes they can be maladaptive. Perhaps, some pro-proliferative oxiforms are enriched in cancer cells. Their probabilistic nature means they may contribute in idiosyncratic ways to certain stochastic elements of the ageing process, especially when entropy may prolong the lifetime of some oxiforms or even favour their formation. Ultimately, the emergent complexity of oxiforms and their potential functional significance is important for understanding redox-regulation in health and disease.
Analysing an entire undigested protein by tandem mass spectrometry (m/s) in top-down proteomics [39,40] can quantify oxiforms [41] by tell-tale chemotype-specific diagnostic mass changes (e.g., PSSG = + 305 Da per modification), which can, in principle, be linked to other proteoforms (e.g., mutated forms or splice variants). Top-down approaches are powerful: Ansong et al. [42] unearthed a chemotype switch from PSSG to PS-L-Cys in salmonella under infection-like conditions. Deconvoluting chemotypes with similar masses, especially when there are many target cysteines, or detecting labile forms (e.g., RSSH) is challenging. Without stable chemical handles, some oxiforms may evolve during the sample processing procedures. Like mobility-shift assays, it is challenging to ascribe top-down redox proteomic detected oxiforms to specific cysteines. Technical advances in individual ion m/s [43] motivated by the human proteoform project, [44] will benefit oxiform research.
The next decade will witness considerable progress when the human proteoform project is completed [25] and gains in chemical diversity and resolution are achieved. [45] Advances in combinatorial chemotype analysis inclusive of orthogonal chemotype-specific and preferably reaction-based chemistry hold the key to unlocking oxiform complexity. Examples include the recent pioneering work from the Kate Carroll [46] and Tobias Dick [47] groups regarding RSOH and RSSH, respectively. Incidentally, the latter used top-down proteomics.
Continued progress in m/s [28] and immunological [48,49] techniques is needed to achieve SC-resolution. SC-resolution can uncover rare oxi- technologies, such as nanopores, may be invaluable. [50] For example, a single-molecule compatible nanopore discerned cationic carrier peptide charged L-cysteine from L-cystine. [51] For now, the prospect of using mobility-shift methods and top-down proteomics (see Figure 3) to answer fundamental questions by quantifying target-specific oxiforms is exciting.
Unravelling the biological roles unique oxiforms play presents formidable technical challenges. Recapitulating their chemistry, especially labile properties like a free radical, and abundance is difficult.
Abundance is important because the functional weight of an emergent property is concentration-dependent. A functional phenotype may only manifest when the number of single-molecules inhabiting the unique oxiform reaches a critical threshold. Promising approaches for functionally phenotyping oxiforms are threefold. First, sophisticated genome editing technologies [52] can mutate multiple cysteine residues to disable certain oxiforms. Second, genetically encoded chemotype mimetics (e.g., an RSOH mimetic amino acid) or introduction handles may recapitulate their chemistry. For example, click chemistry could produce the desired oxiform by selectively adding clickable amino acid mimetics (e.g., a click-in L-Cys-SSH) to distinct residues. Third, computational methods, such as deep learning models and quantum dynamical stimulations, could predict their functional relevance. [53] They are also powerful tools for using established biochemistry (e.g., kinetic constants) [6] to predict the identity of the oxiforms that might exist in a cell and how they might evolve from the initial starting conditions.

CONCLUSION
Humans possessing a similar number of genes to other species, such as worms, argues against an intuitively appealing genomic basis for our complexity [54] and begs the question: what make us so complex?
Some think that much complexity stems from the sheer number of forms a protein molecule can adopt so-called proteoforms. [15]  Oxiforms may contribute to our complexity. Is that why our proteome encodes more cysteine residues than many other species? Potentially.
At any rate, they offer a way to paint different redox pictures on blank single-molecule canvases. The concerted effects, be they subtle

CONFLICT OF INTEREST STATEMENT
The author declares no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to the present article because no new data were generated or analysed.